Combinations of a photosensitizer with a hydrogen sulfide donor, thioredoxin inhibitor or nitroxide for use in photodynamic therapy

ABSTRACT

The invention relates to a combination comprising 
     (i) a compound A comprising a mitochondrial targeting group linked to a group capable of releasing hydrogen sulfide or a pharmaceutically acceptable salt thereof or a prodrug thereof, an inhibitor of the thioredoxin antioxidant system or a pharmaceutically acceptable salt thereof or a prodrug thereof, and/or a nitroxide or a pharmaceutically acceptable salt thereof or a prodrug thereof; and
 
(ii) a photosensitizer or photosensitizer precursor;
 
for use in photodynamic therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 15/315,699, filed Dec. 1, 2016, which is a National Stage ofInternational Patent Application No. PCT/GB2015/051608, filed Jun. 2,2015, which claims benefit of GB 1418676.1, filed Oct. 21, 2014, and GB1409792.7, filed Jun. 2, 2014, the disclosure of each is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to combinations for use in photodynamic therapy,as well as related uses, methods and compositions.

In particular, though not exclusively, the present invention relates toa combination comprising: (i) a compound A comprising a mitochondrialtargeting group linked to a group capable of releasing hydrogen sulfideor a pharmaceutically acceptable salt thereof or a prodrug thereof, aninhibitor of the thioredoxin antioxidant system or a pharmaceuticallyacceptable salt thereof or a prodrug thereof, and/or a nitroxide or apharmaceutically acceptable salt thereof or a prodrug thereof; and (ii)a photosensitizer or photosensitizer precursor; for use in photodynamictherapy. Aspects of the invention relate to the use of this combinationin photodynamic treatment for cosmetic purposes; and to a compositioncomprising components (i) and (ii).

BACKGROUND TO THE INVENTION

Photodynamic therapy (PDT) is a therapy employed routinely in thetreatment of superficial dermatological malignancies and is underinvestigation for a range of additional tumour types. Most applicationsof PDT involve the use of an active compound, known as aphotosensitizer, and a light source, the wavelength of which can bechosen to be appropriate for exciting the photosensitizer to producereactive oxygen species. This leads to the destruction of any tissueswhich have either selectively taken up the photosensitizer or have beenlocally exposed to light.

For example, a PDT treatment of human skin cancer may involve thefollowing steps. Firstly, a photosensitizer precursor is administered tothe patient. The photosensitizer precursor is taken up by the cells andconverted to a photosensitizer. The area to be treated is then exposedto light of the appropriate wavelength. The photosensitizer absorbslight and reacts with nearby tissue oxygen, resulting in reactive oxygenspecies. These reactive oxygen species react with biomolecules, fatallydamaging some of the cells in the treatment area.

PDT has particularly found a niche in the treatment of dermatologicaltumours where light can be readily applied to the surface of the skin;clinically substantial subsets of skin tumours are difficult to treat byconventional therapies (because of size, site or multiple lesionspresentation). In the treatment of skin conditions, the photosensitizeror photosensitizer precursor can be applied topically, and locallyexcited by a light source. In the local treatment of internal cancercells, on the other hand, photosensitizers or photosensitizer precursorscan for example be administered intravenously and light can be deliveredto the target area using endoscopes and fibre optic catheters. Comparedto normal healthy tissues, most types of cancer cells are especiallyactive in both the uptake and accumulation of photosensitizers, whichmakes cancer cells especially vulnerable to PDT, since having morephotosensitizer present in a cell leads to more damage to that cellduring PDT.

Photosensitizer precursors currently employed in dermatological PDTinclude aminolaevulinic acid (ALA), methyl aminolaevulinate (MAL) andhexyl aminolaevulinate (HAL). If ALA, MAL or HAL is used as aphotosensitizer precursor, it is converted by the cells to thephotosensitizer protoporphyrin IX (PpIX).

Porphyrins have long been considered as suitable agents for tumourphotodiagnosis and tumour PDT because cancer cells exhibit asignificantly greater uptake and affinity for porphyrins compared tonormal quiescent tissues; cancer cells therefore naturally accumulateporphyrins. An additional feature of the photosensitizer protoporphyrinIX (PpIX) is its ability to fluoresce, which in combination with cancercells' natural accumulation of porphyrins allows for photodiagnosis (PD)of tumours. PD has been used by surgeons for enabling greater precisionin the removal of tumours, such as for example brain tumours.

PpIX is naturally present in all nucleated mammalian cells at lowconcentrations; it is an intermediate in the biosynthesis of haem. Inthe haem biosynthesis, ALA is converted to PpIX (via a number ofintermediate steps), after which PpIX is converted to haem by theinsertion of a Fe²⁺ ion into PpIX by the enzyme ferrochelatase.

In order for PDT to be effective, it is necessary to increase the amountof PpIX which is present in a cell, which can be achieved by adding moreALA, MAL or HAL to a cell, which will be converted to PpIX. However, thehaem biosynthesis pathway has a maximum limit over which additionalprecursor administration does not produce any additional benefit.Furthermore, excessive ALA oral administration has been demonstrated toinduce liver toxicity in humans. Usually, the presence of free haem actsas a negative feedback mechanism inhibiting ALA synthesis. However, theexogenous administration of large amounts of ALA or MAL bypasses thisnegative feedback signal and results in a temporary accumulation of PpIXwithin the cells, since the insertion of Fe²⁺ into PpIX to form haem isrelatively slow.

Known ways to improve the activity profile in photodynamic therapyinclude limiting the iron supply in a cell by using the iron chelatorCP94, which slows down the step of converting PpIX to haem by insertionof Fe²⁺, allowing PpIX to accumulate in the cell.

A need however remains for other ways to improve the activity profile inphotodynamic therapy, especially since currently photodynamic therapy isnot effective for all tumour types.

STATEMENTS OF THE INVENTION

Aspects of the invention relate to a combination comprising components(i) and (ii) as defined below. The combination may be for use inphotodynamic therapy, or use in photodynamic treatment for cosmeticpurposes. The combination may also be comprised in a composition.

According to a first aspect of the invention there is provided acombination comprising

(i) a compound A comprising a mitochondrial targeting group linked to agroup capable of releasing hydrogen sulfide or a pharmaceuticallyacceptable salt thereof or a prodrug thereof, an inhibitor of thethioredoxin antioxidant system or a pharmaceutically acceptable saltthereof or a prodrug thereof, and/or a nitroxide or a pharmaceuticallyacceptable salt thereof or a prodrug thereof; and(ii) a photosensitizer or photosensitizer precursor;for use in photodynamic therapy.

In an embodiment, the inhibitor of the thioredoxin antioxidant system isa thioredoxin reductase inhibitor or a thioredoxin inhibitor. In anembodiment, the inhibitor of the thioredoxin antioxidant system is athioredoxin reductase inhibitor. In an embodiment, the inhibitor of thethioredoxin antioxidant system is a thioredoxin inhibitor.

For the avoidance of doubt, the term “combination” is used herein tosignify that the components may be provided in any suitable form foradministration to a patient for concurrent or combined action inphotodynamic therapy. Thus the combination may comprise one or morecombined compositions comprising both components (i) and (ii), or a kitcomprising a plurality of compositions together providing the components(i) and (ii). A kit may suitably comprise one or more compositionscomprising one only of the components. In an embodiment, a kit comprisesa first composition comprising component (i) and a second compositioncomprising component (ii). The compositions or components may be of anysuitable form to achieve administration of the components to a patientfor concurrent or combined action in photodynamic therapy. For example,the compositions or components may be adapted for topical, oral,intravenous, intraperitoneal, intradermal or intra-articularadministration.

Using a “combination” comprising component (i) and component (ii) inphotodynamic therapy means that both of the components are administeredto a patient undergoing photodynamic therapy, either simultaneously orsequentially. The components may be administered as part of the samecomposition or separately.

For the avoidance of doubt, use in photodynamic therapy herein meansthat cells are exposed to both components (i) and (ii) after which thosecells are then exposed to light, resulting in at least some of the cellsbeing fatally damaged. In other words, both components (i) and (ii) areused for killing cells on exposure of the cells to light.

In an embodiment, the cells are mammalian cells, such as for examplehuman cells.

In an embodiment, the effect of the photosensitizer or photosensitizerprecursor (component (ii)) on cell death is synergistically enhanced bycomponent (i), which means that the effect of the combination ofcomponents (i) and (ii) on cell death is greater than the sum of theeffects of components (i) and (ii) when they are used individually.

In an embodiment of the first aspect of the invention, the combinationfor use in photodynamic therapy comprises

(i) said compound A comprising a mitochondrial targeting group linked toa group capable of releasing hydrogen sulfide, or a pharmaceuticallyacceptable salt thereof or a prodrug thereof, and

(ii) said photosensitizer or a photosensitizer precursor.

Slow release hydrogen sulfide donors (SRHDs) can be mitochondriallytargeted by linking an H₂S releasing group to a mitochondrial targetinggroup. Mitochondrially targeted SRHDs were previously described in WO2013/045951, which describes investigations into the ability ofnon-targeted SHRD GYY4137 and mitochondrially targeted SRHDs AP39-C10and AP123-C10 to attenuate oxidative stress and damage in human brainmicrovascular endothelial cells (HMEC). WO 2013/045951 showed thatAP39-C10 and GYY4137 inhibit cell death, mitochondrial dysfunction,mitochondrial and cytoplasmic oxidative stress when cells are challengedwith a range of physiological oxidant species, and that mitochondrialtargeting of H₂S confers greater cytoprotection than non-targeted donormolecules. Le Trionnaire S et al., Med. Chem. Commun., 2014, 5, 728-736,and Szczesnya B et al., Nitric Oxide, 2014, DOI:

10.1016/j.niox.2014.04.008 describe that significant protective effectswere observed for GYY4137 as well as AP39-C10 and AP123-C10, withAP39-C10 and AP123-C10 being found significantly more potent. The lossof mitochondrial membrane potential was attenuated by all of the SRHDsinvestigated and the generation of reactive oxygen species (ROS) wasconsiderably decreased.

This embodiment of the first aspect of the invention is concerned withusing SRHDs in conjunction with a pro-oxidant treatment, namelyphotodynamic therapy. As can be seen from Example 1, the inventors havefound that the use of non-targeted H₂S releasing compounds GYY4137,ADT-OH and 4-HTB in combination with MAL and irradiation had nostatistically significant effect on photodynamic cell killing,exhibiting similar levels of cell death compared to MAL only withirradiation. Surprisingly, however, the use of mitochondrially targetedSRHDs AP39-C8, AP39-C10, AP39-C12, AP123-C8, AP123-C10 or AP123-C12 incombination with MAL and irradiation significantly increased cellkilling compared to MAL only with irradiation (see FIGS. 1 and 3).

This observed result runs counter to the vast majority of the researchthat has been carried out investigating H₂S and its role in oxidativestress. For the most part, endogenous H₂S is thought to be responsiblefor maintaining redox homeostasis through scavenging of ONOO⁻, H₂O₂,ClO⁻, O₂ ^(.−) and/or ^(.)NO. By mediating the reduction of thesereactive oxygen species (ROS), H₂S has been shown to prevent theoxidation of proteins and lipoproteins (Whiteman M et al., BiochemBiophys Res Commun, 2006, Apr. 28, 343(1):303-10; Whiteman M et al., JNeurochem., 2004 August, 90(3):765-8; Luo Y et al., Biochem Biophys ResCommun, 2012, 425, 473-7; Whiteman M et al., Biochem Biophys Res Commun,2005, 326, 794-8).

Without wishing to be bound by theory, the observation thatmitochondrially-targeted SRHDs significantly enhanced photodynamic cellkilling may be due to the relatively high concentration of the donorslocalised to the mitochondria. These concentrated donors could releaseconcentrated and localised H₂S, potentially eliciting localised effectsfar more rapidly than the non-targeted alternatives. Although thesemechanisms are not yet elucidated, it may be possible that short-termincubation of cells with mitochondrially targeted SRHD is enough toelicit an inhibition of glucose metabolism, which in turn may sensitisethe cells to photodynamic cell killing.

Without wishing to be bound by theory, another explanation may involvethe potential interaction between the mitochondrially targeted SRHD andirradiation in the presence of a photosensitizer (such as for examplePpIX), which could potentially be inducing photo-dissociation of partsof the H₂S releasing moieties. Experiments carried out in a cell-freesystem (see Example 1 and FIGS. 5 and 6) found that this may beoccurring, as irradiation of AP39-C10 and AP123-C10 in the presence ofphotosensitizer PpIX was found to increase the release of H₂S.

Additionally, without wishing to be bound by theory, the interactionbetween light and hydrogen sulfide in the presence of a photosensitizer(such as for example PpIX) may lead to the generation of polysulfides,thiyl or sulfanyl (HS.) radicals. Additionally or alternatively, it maylead to the generation of persulfides, thiosulfate or sulfite.Polysulfides have been shown to inhibit cell growth and induce apoptosisin cancer cells, which has been highlighted as a possible mechanism ofaction for the anti-cancer properties of garlic extracts (Busch et al.,Int J Oncol, 2010, 36, 743-9). If HS. radicals are formed duringphotodynamic irradiation of a photosensitizer (such as for example PpIX)in the presence of high concentrations of H₂S, it is possible that thiscould lead to cyclical generation of O₂-based radicals such as O₂ ^(.−),leading to an increased generation of damaging ROS, increasing cellkilling.

As can be seen from Example 1, the mitochondrially-targeted SRHDsAP39-C8, AP39-C10, AP39-C12, AP123-C8, AP123-C10 or AP123-C12 provedsurprisingly effective at increasing the efficacy ofphotosensitizer-based photodynamic cell killing, resulting inpredominantly apoptotic cell death.

The compound A is a hydrogen sulfide releasing compound. In particular,the compound A comprises a group that is capable of releasing hydrogensulfide. Typically, the group is capable of releasing hydrogen sulfidein vivo and/or in vitro. In general, the group undergoes a reaction invivo and/or in vitro to generate H₂S and species derived from H₂S underphysiological conditions, such as for example HS⁻, sulfane sulfur,polysulfides, and/or S²⁻. Additionally or alternatively, the group mayundergo a reaction in vivo and/or in vitro to generate thiosulfate orsulfite.

Generally, the group is a moiety capable of releasing hydrogen sulfidethat is linked, either directly or via a linker, to a mitochondrialtargeting group. The mitochondrial targeting group can be attached atany convenient position on the compound that is capable of releasinghydrogen sulfide.

Compounds capable of releasing hydrogen sulfide are well known in theart, see for example G. Caliendo et al (J. Med. Chem., 2010, 53(17),6275-6286). Examples of compounds capable of releasing hydrogen sulfideinclude N-acetyl-penicillamine, S-allyl-cysteine, bucillamine,carbocysteine, cysteamine, cystathionine, homocysteine, mecysteine,methionine, pantetheine, penicilla mine, penicillamine disulfide,thioacetic acid, thiodiglycolic acid, thioglycolic acid, thiolacticacid, 2-thiolhistidine, thiomalic acid, thiosalicylic acid, tiopronin,5-(p-hydroxyphenyl)-3H-1,2-dithiol-3-thione,1,3-dithiol-2-thione-5-carboxylic acid,3-thioxo-3H-1,2-dithiole-5-carboxylic acid and3-thioxo-3H-1,2-dithiole-4-carboxylic acid.

The group capable of releasing hydrogen sulfide can, for example,comprise a sulfide, a disulfide or a polysulfide moiety. Additionally oralternatively, the group capable of releasing hydrogen sulfide can, forexample, comprise a persulfide, a thiosulfate or a perthiol.

In an embodiment, the group capable of releasing hydrogen sulfidecomprises a thiocarbamoyl group, a 5-thioxo-5H-1,2-dithiol-3-yl group, a5-thioxo-5H-1,2-dithiol-4-yl group, a 5-oxo-5H-1,2-dithiol-3-yl group, a5-oxo-5H-1,2-dithiol-4-yl group, a 5-hydroxyimino-5H-1,2-dithiol-3-ylgroup, a 5-hydroxyimino-5H-1,2-dithiol-4-yl group, a phosphinodithioategroup, a phosphinodithioic acid group, a thioketone group, or athioaldehyde group.

In an embodiment, the group capable of releasing hydrogen sulfidecomprises a thiocarbamoyl group or a 5-thioxo-5H-1,2-dithiol-3-yl group.Suitably, the thiocarbamoyl group may form part of a thiobenzamide group(thiobenzamidyl) in the compound A as a whole.

In an embodiment, the group capable of releasing hydrogen sulfide isselected from:

wherein:

-   -   X represents S, O or N—OH;    -   R¹, R² and R³ each independently represent hydrogen, C₁₋₁₂        alkyl, C₁₋₁₂ alkoxy or C₆₋₁₀ aryl, wherein each C₁₋₁₂ alkyl,        C₁₋₁₂ alkoxy or C₆₋₁₀ aryl group is unsubstituted or substituted        by one or more substituents selected from a halogen atom,        hydroxy, C₁₋₁₂ alkoxy, C₁₋₁₂ alkyl, hydroxy-C₁₋₁₂-alkyl,        halo-C₁₋₁₂-alkyl and halo-C₁₋₁₂-alkoxy substituents.

In an embodiment, X is S or O. In an embodiment, X is S.

In an embodiment, R¹, R² and R³ each independently represent hydrogen,C₁₋₁₂ alkyl, or C₁₋₁₂ alkoxy. In an embodiment, R¹, R² and R³ eachindependently represent hydrogen, or C₁₋₁₂ alkyl. In an embodiment, R¹,R² and R³ each independently represent hydrogen.

In an embodiment, the group capable of releasing hydrogen sulfide is:

In an embodiment, the group capable of releasing hydrogen sulfide is:

As can be seen from Example 1, mitochondrially targeted 4-HTBderivatives were found to sensitise A431 cells to photodynamic cellkilling even more than the mitochondrially targeted ADT-OH derivatives.

A mitochondrial targeting group is a group which is capable ofconcentrating the compound in the mitochondria of a cell. For example,following incubation of a cell with a compound comprising amitochondrial targeting group, the concentration of the compound in themitochondria will be higher than the concentration of the compound inthe cytosol. Mitochondrial targeting groups are well known and examplesof appropriate mitochondrial targeting groups are discussed in Souza etal (Mitochondrion 5 (2005) 352-358), Kang et al (The Journal of ClinicalInvestigation, 119, 3, 454-464), Horton et al (Chemistry and Biology 15,375-382), Wang et al (J. Med. Chem., 2007, 50 (21), 5057-5069), Souza etal (Journal of Controlled Release 92 (2003) 189-197), Maiti et al(Angew. Chem. Int. Ed., 2007, 46, 5880-5884), Kanai et al (Org. Biomol.Chem., 2007, 5, 307-309), Senkal et al (J Pharmacol Exp Ther., 317(3),1188-1199), Weiss et al (Proc Natl Acad Sci USA, 84, 5444-5488), Zimmeret al (Br J Pharmacol., 1998, 123(6), 1154-8), Modica-Napolitano et al(Cancer Res., 1996, 56, 544-550), Murphy et al (Ann Rev. Pharm Toxicol.,(2007), 47, 629-656), and Hoye et al (Accounts of Chemical Research, 41,1, 87-97). All of these documents are incorporated by reference. For theavoidance of doubt, all of the mitochondrial targeting groups disclosedin these articles can be used in the compounds comprising amitochondrial targeting group linked to a group capable of releasinghydrogen sulfide.

In an embodiment, the mitochondrial targeting group is a group which iscapable of concentrating the compound specifically in the mitochondrialmatrix of a cell.

In an embodiment, the mitochondrial targeting group is a lipophiliccation or a mitochondrial targeting peptide. In an embodiment, thelipophilic cation is a phosphonium cation, an arsonium cation, anammonium cation, flupritine, MKT-077, a pyridinium ceramide, aquinolium, a liposomal cation, a sorbitol guanidine, a cyclic guanidineor a rhodamine.

Flupritine and MKT-077 are described in Zimmer et al. (Br J Pharmacol.,1998, 123(6), 1154-8) and Modica-Napolitano et al (Cancer Res., 1996,56, 544-550). Mitochondrial targeting peptides are described in Hortonet al (Chemistry and Biology, 2008, 15, 375-382) and Hoye et al(Accounts of Chemical Research, 2008, 41, 1, 87-97).

In an embodiment, the mitochondrial targeting group is a phosphoniumcation. In an embodiment, the phosphonium cation has the formula:

-   -   wherein X₁, X₂ and X₃ each independently represent C₁₋₁₂ alkyl,        C₆₋₁₀ aryl, or C₁₋₁₂alkylene-C₆₋₁₀-aryl, wherein the alkyl and        alkylene groups and moieties are unsubstituted or substituted by        one or more, for example 1, 2 or 3, halogen atoms, hydroxyl,        C₁₋₁₂ alkoxy or halo-C₁₋₁₂-alkoxy groups, and each aryl group or        moiety is unsubstituted or substituted by one, two or three        halogen atoms, hydroxyl, C₁₋₁₂ alkoxy or halo-C₁₋₁₂-alkoxy        groups.

In an embodiment, each alkyl or alkylene group or moiety isunsubstituted or substituted by one or more, such as 1 or 2, halogenatoms. In an embodiment, the alkyl and/or alkylene group or moiety isunsubstituted.

In an embodiment, X₁, X₂ and X₃ are each a C₆₋₁₀ aryl group, for examplea phenyl group. In an embodiment, X₁, X₂ and X₃ are the same.

In an embodiment, the mitochondrial targeting group is atriphenylphosphonium cation (TPP⁺) of the formula:

The group capable of releasing hydrogen sulfide may be linked to one,two, three or more mitochondrial targeting groups. When the groupcapable of releasing hydrogen sulfide is linked to more than onemitochondrial targeting group, each mitochondrial targeting group can bethe same or different. In an embodiment, the group capable of releasinghydrogen sulfide is linked to one mitochondrial targeting group.

In an embodiment, the mitochondrial targeting group is covalently linkedto the group capable of releasing hydrogen sulfide.

The or each mitochondrial targeting group may be linked to the groupcapable of releasing hydrogen sulfide directly or via a linker. Wherethere is more than one mitochondrial targeting group, all of themitochondrial targeting groups may be covalently linked directly to thegroup capable of releasing hydrogen sulfide or all of the mitochondrialtargeting groups may be linked via a linker to the group capable ofreleasing hydrogen sulfide.

In an embodiment, there is one mitochondrial targeting group that islinked via a linker to the group capable of releasing hydrogen sulfide.

The linker may be any moiety capable of linking a mitochondrialtargeting group to the group capable of releasing hydrogen sulfide.

The linker may have a molecular weight of 14 to 1000, such as 28 to 500,for example 44 to 300.

In an embodiment, the linker is a C₁₋₂₀ alkylene which is unsubstitutedor substituted by one or more substituents selected from a halogen atom,hydroxy, C₁₋₁₂ alkoxy, C₁₋₁₂ alkyl, hydroxy-C₁₋₁₂-alkyl,halo-C₁₋₁₂-alkyl and a halo-C₁₋₁₂-alkoxy group, wherein zero or one toten carbon atoms in the alkylene chain are replaced by spacer moietiesselected from C₆₋₁₀ arylene, —O—, —S—, —NR⁴—, —C(O)NR⁴—, —NR⁴C(O)—,—C(O)—, —OC(O)—, —C(O)O— moieties, wherein R⁴ is hydrogen or C₁₋₁₂ alkyland the C₆₋₁₀ arylene moiety is unsubstituted or substituted by one,two, three or four substituents selected from a halogen atom, hydroxy,C₁₋₁₂ alkyl and a C₁₋₁₂ alkoxy group.

In an embodiment, the spacer moieties are selected from C₆₋₁₀ arylene,—O—, —S—, —NR⁴—, —C(O)NR⁴—, —NR⁴C(O)—, —C(O)—, —OC(O)—, —C(O)O—moieties. In an embodiment, the alkylene group consists of 1, 2, 3, 4 or5 spacer moieties. In an embodiment, the alkylene group consists of 1 to3 spacer moieties. In an embodiment, the alkylene group consists of 1 or2 spacer moieties.

In an embodiment, the spacer moieties comprise 0 to 2 C₆₋₁₀ arylene, 0to 2 —S—, 0 to 2 —O—, 0 to 2 —NR⁴—, 0 to 2 —C(O)NR⁴—, 0 to 2 —NR⁴C(O)—,0 to 2 —C(O)—, 0 to 2—OC(O)—, and 0 to 2 —C(O)O— moieties.

In an embodiment, the linker comprises at least one C₆₋₁₀ arylene and atleast one of the —OC(O)— or —C(O)O— spacer moieties.

In an embodiment, the alkylene group is a C₁₋₂₀ alkylene. In anembodiment, the alkylene group is a C₂₋₁₈ alkylene, such as a C₃₋₁₆alkylene.

In an embodiment, the alkylene is a straight chain alkylene.

In an embodiment, the alkylene is unsubstituted or substituted by one ormore, such as 1 or 2, halogen atoms. In an embodiment, said alkylenegroup is unsubstituted.

In an embodiment, the arylene spacer moiety is unsubstituted orsubstituted with one, two or three halogen atoms, hydroxy groups orC₁₋₁₂ alkyl groups. When the arylene spacer moiety carries 2 or moresubstituents, the substituents may be the same or different. In anembodiment, the arylene spacer moiety is unsubstituted.

In an embodiment, the arylene spacer moiety is a phenylene group, whichis unsubstituted or substituted by one, two, three or four substituentsselected from a halogen atom, hydroxy, C₁₋₁₂ alkyl and a C₁₋₁₂ alkoxygroup.

In an embodiment, the linker is represented by the formula:-L′-Y—Z—wherein:

-   -   L′ represents a direct bond or a straight chain C₁₋₂₀ alkylene        group, such as a straight chain C₂₋₁₈ alkylene group, which is        unsubstituted or substituted by one or more substituents        selected from a halogen atom, hydroxy, C₁₋₁₂ alkoxy, C₁₋₁₂        alkyl, hydroxy-C₁₋₁₂-alkyl, halo-C₁₋₁₂-alkyl and a        halo-C₁₋₁₂-alkoxy group;    -   Y represents a direct bond, —OC(O)—, —C(O)O—, —O—, —C(O)NR⁴— or        —NR⁴C(O)— wherein R⁴ is hydrogen or C₁₋₁₂ alkyl;    -   Z represents a direct bond or a phenylene group, which is        unsubstituted or substituted by one, two, three or four        substituents selected from a halogen atom, hydroxy, C₁₋₁₂ alkyl        and a C₁₋₁₂ alkoxy group.

In an embodiment, the alkylene group is unsubstituted or is substitutedwith one, two or three substituents selected from a halogen atom,hydroxy, C₁₋₁₂ alkoxy, C₁₋₁₂ alkyl. In an embodiment, the alkylene groupis unsubstituted.

In an embodiment, L′ is a straight chain alkylene group having theformula:—(CH₂)_(n)—

-   -   wherein n is an integer from 1 to 19.

In an embodiment, n is an integer from 2 to 19. In an embodiment, n isan integer from 2 to 18, from 2 to 17, from 2 to 16, from 2 to 15, from2 to 14, from 2 to 13, from 2 to 12, from 2 to 11, from 3 to 19, from 3to 17, from 3 to 16, from 3 to 15, from 3 to 14, from 3 to 13, from 3 to12, from 3 to 11, from 4 to 19, from 4 to 17, from 4 to 16, from 4 to15, from 4 to 14, from 4 to 13, from 4 to 12, from 4 to 11, from 5 to19, from 5 to 17, from 5 to 16, from 5 to 15, from 5 to 14, from 5 to13, from 5 to 12, from 5 to 11, from 6 to 19, from 6 to 17, from 6 to16, from 6 to 15, from 6 to 14, from 6 to 13, from 6 to 12, from 6 to11, from 7 to 19, from 7 to 17, from 7 to 16, from 7 to 15, from 7 to14, from 7 to 13, from 7 to 12, or from 7 to 11. In an embodiment, n is7, 9, or 11. In an embodiment, n is 11.

In an embodiment, Y is a direct bond, —OC(O)— or —C(O)O—. In anembodiment, Y is —OC(O)— or —C(O)O—. In an embodiment, Y is a —C(O)O—group.

In an embodiment, Z is a phenylene group.

In an embodiment, Z is a para-phenylene group.

In an embodiment, the moiety —Y—Z— has the formula:

In an embodiment, the compound A comprising a mitochondrial targetinggroup linked to a group capable of releasing hydrogen sulfide isrepresented by the formula:MTG-L-Qwherein:

-   -   Q is a group capable of releasing hydrogen sulfide selected        from:

-   -   X represents S, O or N—OH;    -   R¹, R² and R³ each independently represent hydrogen, C₁₋₁₂        alkyl, C₁₋₁₂ alkoxy or C₆₋₁₀ aryl, wherein each C₁₋₁₂ alkyl,        C₁₋₁₂ alkoxy or C₆₋₁₀ aryl group is unsubstituted or substituted        by one or more substituents selected from a halogen atom,        hydroxy, C₁₋₁₂ alkoxy, C₁₋₁₂ alkyl, hydroxy-C₁₋₁₂-alkyl,        halo-C₁₋₁₂-alkyl and halo-C₁₋₁₂-alkoxy substituents;    -   L represents a direct bond or a linker, wherein the linker is a        C₁₋₂₀ alkylene which is unsubstituted or substituted by one or        more substituents selected from a halogen atom, hydroxy, C₁₋₁₂        alkoxy, C₁₋₁₂ alkyl, hydroxy-C₁₋₁₂-alkyl, halo-C₁₋₁₂-alkyl and a        halo-C₁₋₁₂-alkoxy group, wherein zero or one to ten carbon atoms        in the alkylene chain are replaced by spacer moieties selected        from C₆₋₁₀ arylene, —O—, —S—, —NR⁴—, —C(O)NR⁴—, —NR⁴C(O)—,        —C(O)—, —OC(O)—, —C(O)O— moieties, wherein R⁴ is hydrogen or        C₁₋₁₂ alkyl and the C₆₋₁₀ arylene moiety is unsubstituted or        substituted by one, two, three or four substituents selected        from a halogen atom, hydroxy, C₁₋₁₂ alkyl and a C₁₋₁₂ alkoxy        group; and    -   MTG represent a mitochondrial targeting group, such as, for        example, a phosphonium cation;    -   or a pharmaceutically acceptable salt thereof.

In the compounds that have the formula MTG-L-Q, the mitochondrialtargeting group, the linker and the group capable of releasing hydrogensulfide can be as defined above.

In an embodiment, the compound A comprises a cation selected from:

In an embodiment, the compound comprising a mitochondrial targetinggroup linked to a group capable of releasing hydrogen sulfide comprisesa cation having a structure as set out above and an anion that is ahalogen (i.e. F⁻, Cl⁻ or Br⁻). In an embodiment, the anion is a bromideanion.

The compounds comprising a mitochondrial targeting group linked to agroup capable of releasing hydrogen sulfide can, for example, beprepared using the methods described in WO 2013/045951 or by routinemodifications thereof, or by using conventional methods known in theart.

In an embodiment of the first aspect of the invention, the combinationfor use in photodynamic therapy comprises

(i) said inhibitor of the thioredoxin antioxidant system, or apharmaceutically acceptable salt thereof or a prodrug thereof, and

(ii) said photosensitizer or photosensitizer precursor.

The term “inhibitors of the thioredoxin antioxidant system” comprisesinhibitors of thioredoxin (Trx) and inhibitors of thioredoxin reductase.

In an embodiment, the inhibitor of the thioredoxin antioxidant system isa thioredoxin reductase inhibitor or a thioredoxin inhibitor.

In an embodiment, the inhibitor of the thioredoxin antioxidant system isa thioredoxin reductase inhibitor.

Thioredoxin reductase forms part of the thioredoxin antioxidant system;thioredoxin reductase enzymatically reduces the active site disulfide(S₂) of oxidised thioredoxin to a dithiol (SH₂).

Compounds which are known to inhibit thioredoxin reductase are calledthioredoxin reductase inhibitors. Evidence has shown that lowconcentrations of some thioredoxin reductase inhibitors can effectivelyinhibit the thioredoxin system whilst maintaining cell viability (Cai etal., Free Radic Biol Med, 2012, 52, 257-65).

This embodiment of the first aspect of the invention is concerned withusing thioredoxin reductase inhibitors in conjunction with a pro-oxidanttreatment, namely photodynamic therapy. As can be seen from Example 2,the inventors have found that surprisingly, the use of a thioredoxinreductase inhibitor in combination with a photosensitizer precursor andirradiation significantly increased cell killing compared to aphotosensitizer precursor only with irradiation (see FIG. 9).

In an embodiment, the thioredoxin reductase inhibitor is agold-containing compound.

In an embodiment, the gold-containing compound comprises a gold atomlinked to a sulfur atom.

In an embodiment, the gold-containing compound comprises a gold(I)complex.

In an embodiment, the gold-containing compound comprises a gold(I)thiolate. In an embodiment, the gold-containing compound comprisesauranofin, aurothiomalate, aurothiosulfate, and/or aurothioglucose.

In an embodiment, the gold-containing compound is auranofin.

Auranofin (3,4,5-triacetyloxy-6-(acetyloxymethyl) oxane-2-thiolate;triethylphosphanium) is a potent thioredoxin reductase inhibitor. Theorganic gold compound was originally used to treat rheumatoid arthritis,effectively decreasing blood IgG levels and joint swelling (Finkelstein,A. E. et al., Annals of the Rheumatic Diseases, 1976, 35, 251-7).Inhibition of thioredoxin reductase by auranofin is thought to be due toits high reactivity with the active site selenocysteine, a property thatis shared by many gold-containing compounds (Gromer, S. et al., J BiolChem, 1998, 273, 20096-101).

In an embodiment, the gold-containing compound is sodium aurothiomalateor sodium aurothiosulfate.

In an embodiment, the gold-containing compound is aurothioglucose.Aurothioglucose is thought to exist as a polymer, but the polymerisedstructure has not yet been established. Speculative structures ofaurothioglucose include:

In an embodiment, the thioredoxin reductase inhibitor is DNCB.

DNCB (1-chloro-2,4-dinitrobenzene) is an alkylating agent, often used todeplete intracellular glutathione (GSH) levels, that was discovered toirreversibly inactivate thioredoxin reductase at low concentrations.Furthermore, this inactivation effect is in the region of 10,000-foldfaster than the alkylation of GSH (Arner, E. S. et al., J Biol Chem,1995, 270, 3479-82). Complete inactivation of 50 nM reduced thioredoxinreductase was achieved after 5 minutes incubation with 100 μM DNCB. Thisinactivation only occurred in the presence of NAD(P)H and persisted uponremoval of DNCB. This persistence led to the theory that DNCB mediatedinactivation of thioredoxin reductase was induced by alkylation of theactive site thiols.

In an embodiment, the inhibitor of the thioredoxin antioxidant system isa thioredoxin inhibitor.

In an embodiment, the thioredoxin inhibitor is PX12.

In an embodiment of the first aspect of the invention, the combinationfor use in photodynamic therapy comprises

(i) said nitroxide, or a pharmaceutically acceptable salt thereof or aprodrug thereof, and

(ii) said photosensitizer or photosensitizer precursor.

Nitroxides are known to be effective antioxidants which can protectagainst oxidative damage. For example, the nitroxide TEMPOL(4-hydroxy-2,2,6,6-tetra methylpiperidine 1-oxyl) has been studied as aprotective agent against pro-oxidant doxorubicin-induced cardiactoxicity (Monti, E. et al., Free Radic Biol Med, 1996, 21, 463-70). Inisolated rat hearts, TEMPOL was shown to significantly decrease thecontractile impairment associated with doxorubicin toxicity. This wasreflected by a decrease in oxidative damage, primarily lipidperoxidation. Derivatives of TEMPOL, OT-551 and OT-674 have also shownpromise in the protection of light-induced retinal photoreceptor damage(Tanito, M. et al., Invest Ophthalmol Vis Sci, 2007, 48, 1900-5). Ratsthat had been treated with H₂O, OT-551 or OT-674 were exposed to 2700lux white light for 6 hours. Retinal damage was evaluatedhistopathologically 5-7 days after exposure. Rats treated with H₂O lostelectroretinogram b-wave amplitudes and the retinal outer nuclear layerthickness was significantly decreased. Additionally, increased levels of4-HNE were observed, which is known to cause lipid peroxidation andprotein modification. TEMPOL derivatives OT-551 and OT-674 thereforesignificantly protected against light-induced damage in a dose-dependentmanner and at the highest dose, OT-551 completely inhibited 4-HNEmediated protein modification.

This embodiment of the first aspect of the invention is concerned withusing nitroxides in conjunction with a pro-oxidant treatment, namelyphotodynamic therapy. Despite the nitroxides' well-known protectiveeffects, as can be seen from Example 3 the inventors have surprisinglyfound that the use of a nitroxide in combination with a photosensitizerprecursor and irradiation did not have an inhibitory effect onphotodynamic cell killing but instead significantly increased cell deathcompared to a photosensitizer precursor alone with irradiation (see FIG.12).

Nitroxides can exist in three different oxidation states, namely in thenitroxide, oxoammonium cation or hydroxylamine form. The ability ofnitroxides to efficiently undergo one-electron reductions has provenuseful in the dismutation of O₂ ^(.−) and the reduction process is saidto be SOD-mimetic (where SOD stands for superoxide dismutase), in thatit is a catalytic reaction yielding the same products as SOD-mediateddismutation of O₂ ^(.−) (Krishna, M. C. et al., Proc Natl Acad Sci USA,1992, 89, 5537-41). For example, shown below are the redoxtransformations between the nitroxide (top), oxoammonium cation (left)and hydroxylamine (right) oxidation states of TEMPO(2,2,6,6-tetramethyl-1-piperidinyloxy) and TEMPOL (4-hydroxy-TEMPO or4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl), which are stable freeradical cyclic nitroxides known to possess SOD-mimetic properties.

The SOD-mimetic properties are thought to contribute to the nitroxides'well-documented protective effects against oxidative damage.

Despite the nitroxides' well-known protective effects, as can be seenfrom Example 3, nitroxides proved surprisingly effective at increasingthe efficacy of photosensitizer-based photodynamic cell killing.

In an embodiment, the nitroxide is optionally substituted TEMPO.

In an embodiment, the nitroxide is TEMPO optionally substituted in the4-position.

In an embodiment, the nitroxide is a compound of the formula:

-   -   wherein    -   R¹⁰ and R¹¹ are independently selected from —H, —OR¹²,        —OC(O)R¹², —C(O)OR¹², —C(O)R¹², —NR¹²R¹³, —C(O)NR¹²R¹³,        —NR¹²C(O)R¹³, —NCS, optionally substituted alkyl, optionally        substituted cycloalkyl, optionally substituted alkenyl,        optionally substituted alkynyl, optionally substituted aryl,        optionally substituted heteroaryl, optionally substituted        heterocyclyl, or a protecting group, or R¹⁰ and R¹¹ may be        joined together to form part of an optionally substituted cyclic        group; and    -   R¹² and R¹³ are independently selected from —H, optionally        substituted alkyl, optionally substituted cycloalkyl, optionally        substituted alkenyl, optionally substituted alkynyl, optionally        substituted aryl, optionally substituted heteroaryl, optionally        substituted heterocyclyl, or alkoxyl, or R¹² and R¹³ may be        joined together to form part of an optionally substituted cyclic        group.

In an embodiment, at least one of R¹⁰ and R¹¹ is —H.

In an embodiment, the nitroxide is any one of the following compounds:

In an embodiment, the nitroxide is TEMPO or TEMPOL.

In an embodiment, the combination for use in photodynamic therapycomprises a prodrug of the nitroxide. In an embodiment, the prodrug is anitrone.

In an embodiment of the first aspect of the invention, thephotosensitizer or photosensitizer precursor is a photosensitizerprecursor. In an embodiment, the photosensitizer precursor comprises aprecursor of protoporphyrin IX (PpIX). In an embodiment, thephotosensitizer precursor comprises aminolaevulinic acid (ALA), methylaminolaevulinate (MAL), and/or hexyl aminolaevulinate (HAL). In anembodiment, the photosensitizer precursor comprises methylaminolaevulinate (MAL).

In an embodiment, the photosensitizer or photosensitizer precursor is aphotosensitizer. In an embodiment, the photosensitizer comprisesprotoporphyrin IX (PpIX).

In an embodiment of the first aspect of the invention, the combinationcomprising the components (i) and (ii) is for use in treating a medicalcondition which is responsive to photodynamic therapy. In an embodiment,the combination is for use in treating a condition, which is caused byand/or exacerbated by the abnormal proliferation of cells, byphotodynamic therapy. In an embodiment, the combination is for use intreating cancer, by photodynamic therapy.

In an embodiment, the combination comprising the components (i) and (ii)is for use in treating scleroderma, lichen sclerosus, psoriasis orwarts, by photodynamic therapy. In an embodiment, the combination is foruse in treating chronic wounds, by photodynamic therapy. Such chronicwounds may, for example, be leg ulcers in the elderly. In an embodiment,the combination is for use in treating acne, by photodynamic therapy. Inan embodiment, the combination is for use in treating a microbialinfection, by photodynamic therapy. Such a microbial infection may, forexample, be caused by bacteria, fungi, viruses and/or yeasts. In anembodiment, the combination is for use in treating a parasiticinfestation, by photodynamic therapy. In an embodiment, the combinationis for use in treating rheumatoid arthritis, by photodynamic therapy. Inan embodiment, the combination is for use in bone marrow purging, byphotodynamic therapy, in the treatment of leukaemia. In an embodiment,the combination is for use in treating pulmonary fibrosis, byphotodynamic therapy. In an embodiment, the combination is for use intreating restenosis, by photodynamic therapy.

In an embodiment, the combination comprising the components (i) and (ii)for use according to the first aspect of the invention is administeredtopically. In an embodiment, the combination is administered orally. Inan embodiment, the combination is administered intravenously. In anembodiment, the combination is administered intraperitoneally. In anembodiment, the combination is administered intradermally. In anembodiment, the combination is administered intra-articularly.

According to a second aspect of the invention there is provided the useof a combination comprising (i) a compound A comprising a mitochondrialtargeting group linked to a group capable of releasing hydrogen sulfideor a pharmaceutically acceptable salt thereof or a prodrug thereof, aninhibitor of the thioredoxin antioxidant system or a pharmaceuticallyacceptable salt thereof or a prodrug thereof, and/or a nitroxide or apharmaceutically acceptable salt thereof or a prodrug thereof; and (ii)a photosensitizer or photosensitizer precursor, in photodynamictreatment for cosmetic purposes.

In an embodiment, the inhibitor of the thioredoxin antioxidant system isa thioredoxin reductase inhibitor or a thioredoxin inhibitor. In anembodiment, the inhibitor of the thioredoxin antioxidant system is athioredoxin reductase inhibitor. In an embodiment, the inhibitor of thethioredoxin antioxidant system is a thioredoxin inhibitor.

In an embodiment of the second aspect of the invention, the combinationcomprises (i) the compound A comprising a mitochondrial targeting grouplinked to a group capable of releasing hydrogen sulfide, or apharmaceutically acceptable salt thereof or a prodrug thereof, and (ii)the photosensitizer or photosensitizer precursor. In an embodiment, thecompound A is as described for the first aspect of the invention and/orthe photosensitizer or photosensitizer precursor is as described for thefirst aspect of the invention.

In an embodiment of the second aspect of the invention, the combinationcomprises (i) the inhibitor of the thioredoxin antioxidant system, or apharmaceutically acceptable salt thereof or a prodrug thereof, and (ii)the photosensitizer or photosensitizer precursor. In an embodiment, theinhibitor of the thioredoxin antioxidant system is as described for thefirst aspect of the invention and/or the photosensitizer orphotosensitizer precursor is as described for the first aspect of theinvention.

In an embodiment of the second aspect of the invention, the combinationcomprises (i) the nitroxide, or a pharmaceutically acceptable saltthereof or a prodrug thereof, and (ii) the photosensitizer orphotosensitizer precursor. In an embodiment, the nitroxide is asdescribed for the first aspect of the invention and/or thephotosensitizer or photosensitizer precursor is as described for thefirst aspect of the invention.

In an embodiment of the second aspect of the invention, the combinationis used in the photodynamic treatment for cosmetic purposes ofhypertrophic scars, acne scars, wrinkles (rhytides), actinically damagedskin (also known as photodamaged skin or sun damaged skin), rosacea,actinic keratosis, sebaceous gland hyperplasia, lentigines, hirsutism,telangiectasias, port wine stains, erythema, poikiloderma, melisma,dyschromia, hyperpigmentation, mottled or blotchy pigmentation, roughskin patches, poor skin texture, enlarged pores, and/or skin laxity. Inan embodiment, the combination is used in cosmetic photorejuvenation ofskin by photodynamic treatment.

According to a third aspect of the invention there is provided a methodof treatment of a human or animal patient suffering from or at risk ofsuffering from a condition which is caused by and/or exacerbated by theabnormal proliferation of cells, the method involving administering tothe patient a therapeutically effective amount of a combinationcomprising (i) a compound A comprising a mitochondrial targeting grouplinked to a group capable of releasing hydrogen sulfide or apharmaceutically acceptable salt thereof or a prodrug thereof, aninhibitor of the thioredoxin antioxidant system or a pharmaceuticallyacceptable salt thereof or a prodrug thereof, and/or a nitroxide or apharmaceutically acceptable salt thereof or a prodrug thereof; and (ii)a photosensitizer or photosensitizer precursor; and exposing a region ofthe patient containing the combination to light as part of aphotodynamic therapy.

In an embodiment, the inhibitor of the thioredoxin antioxidant system isa thioredoxin reductase inhibitor or a thioredoxin inhibitor. In anembodiment, the inhibitor of the thioredoxin antioxidant system is athioredoxin reductase inhibitor. In an embodiment, the inhibitor of thethioredoxin antioxidant system is a thioredoxin inhibitor.

In an embodiment of the third aspect of the invention, the combinationcomprises (i) the compound A comprising a mitochondrial targeting grouplinked to a group capable of releasing hydrogen sulfide, or apharmaceutically acceptable salt thereof or a prodrug thereof, and (ii)the photosensitizer or photosensitizer precursor. In an embodiment, thecompound A is as described for the first aspect of the invention and/orthe photosensitizer or photosensitizer precursor is as described for thefirst aspect of the invention.

In an embodiment of the third aspect of the invention, the combinationcomprises (i) the inhibitor of the thioredoxin antioxidant system, or apharmaceutically acceptable salt thereof or a prodrug thereof, and (ii)the photosensitizer or photosensitizer precursor. In an embodiment, theinhibitor of the thioredoxin antioxidant system is as described for thefirst aspect of the invention and/or the photosensitizer orphotosensitizer precursor is as described for the first aspect of theinvention.

In an embodiment of the third aspect of the invention, the combinationcomprises (i) the nitroxide, or a pharmaceutically acceptable saltthereof or a prodrug thereof, and (ii) the photosensitizer orphotosensitizer precursor. In an embodiment, the nitroxide is asdescribed for the first aspect of the invention and/or thephotosensitizer or photosensitizer precursor is as described for thefirst aspect of the invention.

According to a fourth aspect of the invention there is provided acomposition comprising (i) a compound A comprising a mitochondrialtargeting group linked to a group capable of releasing hydrogen sulfideor a pharmaceutically acceptable salt thereof or a prodrug thereof, aninhibitor of the thioredoxin antioxidant system or a pharmaceuticallyacceptable salt thereof or a prodrug thereof, and/or a nitroxide or apharmaceutically acceptable salt thereof or a prodrug thereof; and (ii)a photosensitizer or photosensitizer precursor.

In an embodiment, the inhibitor of the thioredoxin antioxidant system isa thioredoxin reductase inhibitor or a thioredoxin inhibitor. In anembodiment, the inhibitor of the thioredoxin antioxidant system is athioredoxin reductase inhibitor. In an embodiment, the inhibitor of thethioredoxin antioxidant system is a thioredoxin inhibitor.

In an embodiment of the fourth aspect of the invention, the combinationcomprises (i) the compound A comprising a mitochondrial targeting grouplinked to a group capable of releasing hydrogen sulfide, or apharmaceutically acceptable salt thereof or a prodrug thereof, and (ii)the photosensitizer or photosensitizer precursor. In an embodiment, thecompound A is as described for the first aspect of the invention and/orthe photosensitizer or photosensitizer precursor is as described for thefirst aspect of the invention.

In an embodiment of the fourth aspect of the invention, the combinationcomprises (i) the inhibitor of the thioredoxin antioxidant system, or apharmaceutically acceptable salt thereof or a prodrug thereof, and (ii)the photosensitizer or photosensitizer precursor. In an embodiment, theinhibitor of the thioredoxin antioxidant system is as described for thefirst aspect of the invention and/or the photosensitizer orphotosensitizer precursor is as described for the first aspect of theinvention.

In an embodiment of the fourth aspect of the invention, the combinationcomprises (i) the nitroxide, or a pharmaceutically acceptable saltthereof or a prodrug thereof, and (ii) the photosensitizer orphotosensitizer precursor. In an embodiment, the nitroxide is asdescribed for the first aspect of the invention and/or thephotosensitizer or photosensitizer precursor is as described for thefirst aspect of the invention.

In an embodiment of the fourth aspect of the invention, the compositionis a pharmaceutical composition which further comprises apharmaceutically acceptable carrier. In an embodiment, components (i)and (ii) are present in the composition in a pharmaceutically effectiveamount. Throughout this specification, the term “pharmaceutical”includes veterinary. In an embodiment, the composition is a topical skintreatment formulation.

According to a fifth aspect of the invention there is provided acompound A comprising a mitochondrial targeting group linked to a groupcapable of releasing hydrogen sulfide or a pharmaceutically acceptablesalt thereof or a prodrug thereof, for use in photodynamic therapy forthe purpose of enhancing cell death, wherein the photodynamic therapycomprises the use of a combination of the compound A or apharmaceutically acceptable salt thereof or a prodrug thereof and aphotosensitizer or a photosensitizer precursor. In an embodiment, thecompound A is as described for the first aspect of the invention and/orthe photosensitizer or photosensitizer precursor is as described for thefirst aspect of the invention.

According to a sixth aspect of the invention there is provided aninhibitor of the thioredoxin antioxidant system or a pharmaceuticallyacceptable salt thereof or a prodrug thereof, for use in photodynamictherapy for the purpose of enhancing cell death, wherein thephotodynamic therapy comprises the use of a combination of the inhibitorof the thioredoxin antioxidant system or a pharmaceutically acceptablesalt thereof or a prodrug thereof and a photosensitizer or aphotosensitizer precursor. In an embodiment, the inhibitor of thethioredoxin antioxidant system is as described for the first aspect ofthe invention and/or the photosensitizer or photosensitizer precursor isas described for the first aspect of the invention.

In an embodiment, the inhibitor of the thioredoxin antioxidant system isa thioredoxin reductase inhibitor or a thioredoxin inhibitor. In anembodiment, the inhibitor of the thioredoxin antioxidant system is athioredoxin reductase inhibitor. In an embodiment, the inhibitor of thethioredoxin antioxidant system is a thioredoxin inhibitor.

According to a seventh aspect of the invention there is provided anitroxide or a pharmaceutically acceptable salt thereof or a prodrugthereof, for use in photodynamic therapy for the purpose of enhancingcell death, wherein the photodynamic therapy comprises the use of acombination of the nitroxide or a pharmaceutically acceptable saltthereof or a prodrug thereof and a photosensitizer or a photosensitizerprecursor. In an embodiment, the nitroxide is as described for the firstaspect of the invention and/or the photosensitizer or photosensitizerprecursor is as described for the first aspect of the invention.

For the avoidance of doubt, in the second, third, fourth, fifth, sixthand seventh aspects of the invention, the compound A, the inhibitor ofthe thioredoxin antioxidant system, the nitroxide and/or thephotosensitizer or photosensitizer precursor can be as defined above forthe first aspect of the invention.

Definitions

It is to be understood that the wavy line in any chemical structures ormoieties represented herein, such as shown below, indicates the point ofattachment of that structure or moiety.

Any reference to groups or compounds for “releasing” or that are capableof “releasing” hydrogen sulfide as used herein refers to a group or acompound that undergoes a chemical reaction, e.g. in vivo and/or invitro, to produce H₂S, HS⁻, S²⁻ and/or further derived species such asfor example sulfane sulfur and/or polysulfides. Additionally oralternatively, the group may undergo a reaction in vivo and/or in vitroto generate thiosulfate or sulfite. In aqueous solution, H₂S dissociatesto form two dissociation states; the hydrosulfide anion (HS⁻) and thesulfide anion (S²⁻). The group or compound may therefore produce H₂S,HS⁻, S²⁻, sulfane sulfur and/or polysulfides, depending on thesurrounding physiological conditions. Additionally or alternatively, thegroup may produce thiosulfate or sulfite.

The compound A comprising a mitochondrial targeting group linked to agroup capable of releasing hydrogen sulfide and/or the inhibitor of thethioredoxin antioxidant system (for example a thioredoxin reductaseinhibitor) may be present in the form of a pharmaceutically acceptablesalt. Prodrugs of the compound A comprising a mitochondrial targetinggroup linked to a group capable of releasing hydrogen sulfide, theinhibitor of the thioredoxin antioxidant system (for example athioredoxin reductase inhibitor) and/or the nitroxide may also bepresent in the form of a pharmaceutically acceptable salt. For use inpharmaceutical compositions, the salts of the compounds refer tonon-toxic “pharmaceutically acceptable salts”. Examples ofpharmaceutically acceptable salts are discussed in Berge et al (J.Pharm. Sci., 1977, 66, 1-19).

Pharmaceutically acceptable salt forms include pharmaceuticallyacceptable acidic/anionic or basic/cationic salts.

Examples of pharmaceutically acceptable acidic/anionic salts includeacetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide,calcium edetate, camsylate, carbonate, chloride, citrate,dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, malonate, mandelate, mesylate,methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, hydrogensulfate, tannate, tartrate,teoclate, tosylate, and triethiodide salts.

Examples of pharmaceutically acceptable basic/cationic salts includesodium, potassium, calcium, magnesium, diethanolamine,N-methyl-D-glucamine, L-lysine, L-arginine, ammonium, ethanolamine,piperazine and triethanolamine salts.

If the compound is anionic, or has a functional group which may beanionic, then a salt may be formed with a suitable cation. Examples ofsuitable inorganic cations include alkali metal ions, such as Na⁺ andK⁺, alkaline earth cations, such as Ca²⁺ and Mg²⁺, and other cationssuch as Al³⁺. Examples of suitable organic cations include ammonium ion(i.e., NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺, NHR₃⁺, NR₄ ⁺, where R is an alkyl group).

If the compound is cationic, or has a functional group which may becationic, then a salt may be formed with a suitable anion. Examples ofsuitable inorganic anions include those derived from the followinginorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric,sulfurous, nitric, nitrous, phosphoric, and phosphorous. Examples ofsuitable organic anions include those derived from the following organicacids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic,camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic,ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic,hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic,lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic,oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic,propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric,toluenesulfonic, and valeric.

If the compound has both a cationic functional group, or a functionalgroup that can become cationic, and an anionic functional group, or afunctional group that can become anionic, then the compound may bepresent as a zwitterion.

The compound A comprising a mitochondrial targeting group linked to agroup capable of releasing hydrogen sulfide, the inhibitor of thethioredoxin antioxidant system (for example a thioredoxin reductaseinhibitor) and/or the nitroxide may be present in the form of a prodrug.A prodrug of a compound is a form of the compound which can be convertedto the compound in vivo, for example through a normal metabolic process.

The term “hydrogen” or “hydrogen atom” as used herein refers to a —Hmoiety.

The term “halogen” or “halogen atom” as used herein refers to a —F, —Cl,—Br or —I moiety.

The term “hydroxy” as used herein refers to an —OH moiety.

The term “alkyl” as used herein refers to a monovalent moiety obtainedby removing a hydrogen atom from a carbon atom of a hydrocarbon compoundhaving from 1 to 12 carbon atoms (unless otherwise specified), which maybe aliphatic or alicyclic, which may be saturated or unsaturated (e.g.partially unsaturated or fully unsaturated), and which may be linear orbranched. Thus, the term “alkyl” includes the sub-classes alkenyl,alkynyl, cycloalkyl, cycloalkenyl and cycloalkynyl below.

In the context of alkyl groups, the prefix C₁₋₁₂ denotes the number ofcarbon atoms, or range of number of carbon atoms present in that group.Thus, the term “C₁₋₁₂ alkyl” refers to an alkyl group having from 1 to12 carbon atoms. The first prefix may vary according to the nature ofthe alkyl group. Thus, if the alkyl group is an alkenyl or alkynylgroup, then the first prefix must be at least 2 (e.g. C₂₋₁₂). For cyclic(e.g. cycloalkyl, cycloalkenyl, cycloalkynyl) or branched alkyl groups,the first prefix must be at least 3 (e.g. C₃₋₁₂).

Examples of saturated alkyl groups include methyl (C₁), ethyl (C₂),propyl (C₃), butyl (C₄), pentyl (C₅), hexyl (C₆), heptyl (C₇), octyl(C₈), nonyl (C₉) and decyl (C₁₀). Examples of saturated linear alkylgroups include, but are not limited to, methyl (C₁), ethyl (C₂),n-propyl (C₃), n-butyl (C₄), n-pentyl (amyl) (C₅), n-hexyl (C₆), andn-heptyl (C₇). Examples of saturated branched alkyl groups includeiso-propyl (C₃), iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄),iso-pentyl (C₅), and neo-pentyl (C₅).

The term “alkenyl” refers to an alkyl group having one or morecarbon-carbon double bonds. Examples of unsaturated alkenyl groupsinclude ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃) and 2-propenyl(allyl, —CH—CH═CH₂).

The term “alkynyl” refers to an alkyl group having one or morecarbon-carbon triple bonds. Examples of unsaturated alkynyl groupsinclude, but are not limited to, ethynyl (ethinyl, —C≡CH) and 2-propynyl(propargyl, —CH₂—C≡CH).

The term “cycloalkyl” refers an alkyl group which is also a cyclylgroup; that is, a monovalent moiety obtained by removing a hydrogen atomfrom an alicyclic ring atom of a carbocyclic compound (i.e. a compoundwhere all of the ring atoms are carbon atoms). The ring may be saturatedor unsaturated (e.g. partially unsaturated or fully unsaturated), whichmoiety has from 3 to 12 carbon atoms (unless otherwise specified). Thus,the term “cycloalkyl” includes the sub-classes cycloalkenyl andcycloalkynyl. In an embodiment, each ring has from 3 to 7 ring carbonatoms. Examples of cycloalkyl groups include those derived from (i)saturated monocyclic hydrocarbon compounds: cyclopropane (C₃),cyclobutane (C₄), cyclopentane (C₅), cyclohexane (C₆), cycloheptane (C₇)and methylcyclopropane (C₄); (ii) unsaturated monocyclic hydrocarboncompounds: cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅),cyclohexene (C₆), methylcyclopropene (C₄) and dimethylcyclopropene (C₅);(iii) saturated polycyclic hydrocarbon compounds: thujane (C₁₀), carane(C₁₀), pinane (C₁₀), bornane (C₁₀), norcarane (C₇), norpinane (C₇),norbornane (C₇), adamantane (C₁₀), decalin (C₁₀); (iv) unsaturatedpolycyclic hydrocarbon compounds: camphene (C₁₀), limonene (C₁₀), pinene(C₁₀); and (v) polycyclic hydrocarbon compounds having an aromatic ring:indene (C₉), indane (C₉) and tetraline (C₁₀).

In an embodiment, a reference to an alkyl group described herein is aC₁₋₁₂ alkyl group, such as a C₁₋₈ alkyl group, for example a C₁₋₆ alkylgroup, or a C₁₋₄ alkyl group. The alkyl groups in the invention can besaturated alkyl groups or saturated cycloalkyl groups, for examplesaturated, unbranched alkyl groups.

The phrase “optionally substituted” as used herein refers to a parentgroup which may be unsubstituted or which may be substituted with one ormore, for example one or two, substituents. The substituents on an“optionally substituted” group may for example be selected from alkyl,cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl groups;carboxylic acids and carboxylate ions; carboxylate esters; carbamates;alkoxyl groups; ketone and aldehyde groups; amine and amide groups; —OH;—CN; —NO₂; and halogens.

The term “substituents” is used herein in the conventional sense andrefers to a chemical moiety, which is covalently attached to, or ifappropriate, fused to, a parent group.

The term “aryl” as used herein refers to a monovalent moiety obtained byremoving a hydrogen atom from an aromatic ring atom of an aromaticcompound, which moiety has from 6 to 10 ring carbon atoms (unlessotherwise specified). In an embodiment, the aryl group is a phenylgroup.

The term “heteroaryl” as used herein refers to a monovalent moietyobtained by removing a hydrogen atom from a heteroaromatic compound,which moiety may for example be a monocyclic or bicyclic group. Theheteroaryl moiety may contain from 1 to 12 carbon atoms (unlessotherwise specified) and one or more N, O or S atoms. The heteroarylmoiety may be a 5 or 6-membered ring containing one or more N atoms.

The term “heterocyclyl” as used herein refers to a monovalent moietyobtained by removing a hydrogen atom from a ring atom of a heterocycliccompound, which moiety may for example be a monocyclic or bicyclicgroup. The heterocyclyl group may contain from 1 to 12 carbon atoms(unless otherwise specified) and one or more N, O or S atoms.

The term “alkoxy” used herein refers to an alkyl-oxy group, where thealkyl group is as defined above and has from 1 to 12 carbon atoms(unless otherwise specified). In an embodiment, the alkyl moiety in analkoxy group is a saturated alkyl group or a saturated cycloalkyl group.In an embodiment, the alkyl moiety is a saturated, unbranched alkylgroup. Examples of C₁₋₁₂ alkoxy groups include —OMe (methoxy), —OEt(ethoxy), —O(^(n)Pr) (n-propoxy), —O(^(i)Pr) (isopropoxy), —O(^(n)Bu)(n-butoxy), —O(^(s)Bu) (sec-butoxy), —O(^(i)Bu) (isobutoxy), and—O(^(t)Bu) (tert-butoxy).

The term “alkylene” as used herein refers to a bidentate moiety obtainedby removing two hydrogen atoms, either both from the same carbon atom,or one from each of two different carbon atoms, of a hydrocarboncompound having from 1 to 20 carbon atoms (unless otherwise specified),which may be aliphatic or alicyclic, and which may be saturated,partially unsaturated, or fully unsaturated. Thus, the term “alkylene”includes the sub-classes alkenylene, alkynylene, cycloalkylene asdiscussed below. The prefix (e.g. C₁₋₄, C₁₋₇, C₁₋₂₀) denotes the numberof carbon atoms, or a range for the number of carbon atoms. For example,the term “C₁₋₂₀alkylene” used herein, refers to an alkylene group havingfrom 1 to 20 carbon atoms.

Examples of linear saturated C₁₋₂₀alkylene groups include —(CH₂)_(n)—where n is an integer from 1 to 20, such as —CH₂— (methylene), —CH₂CH₂—(ethylene), —CH₂CH₂CH₂— (propylene), and —CH₂CH₂CH₂CH₂— (butylene).Examples of branched saturated C₁₋₂₀alkylene groups include —CH(CH₃)—,—CH(CH₃)CH₂—, and —CH(CH₃)CH₂CH₂—. Examples of linear partiallyunsaturated C₂₋₂₀alkylene groups include —CH═CH— (vinylene), —CH═CHCH₂—,—CH₂—CH═CH₂—, and —CH═CHCH₂CH₂—. Examples of branched partiallyunsaturated C₁₋₂₀alkylene groups include —C(CH₃)═CH—, —C(CH₃)═CHCH₂— and—CH═CHCH(CH₃)—. Examples of alicyclic saturated C₃₋₂₀alkylene groupsinclude cyclopentylene (e.g. cyclopent-1,3-ylene) and cyclohexylene(e.g. cyclohex 1,4 ylene). Examples of alicyclic partially unsaturatedC₂₋₂₀alkylene groups include cyclopentenylene (e.g.4-cyclopenten-1,3-ylene), cyclohexenylene (e.g. 2 cyclohexen-1,4-ylene;3 cyclohexen-1,2-ylene; 2,5 cyclohexadien-1,4-ylene).

In an embodiment, a reference to an alkylene group described herein is aC₁₋₂₀alkylene group, such as a C₁₋₁₂alkylene group, for example aC₂₋₈alkylene group, or a C₃₋₇alkylene group. In an embodiment, thealkylene groups can be saturated alkyl groups or saturated cycloalkylgroups, such as saturated, unbranched alkyl groups (i.e. straight chainalkylene group).

The term “arylene” as used herein refers to a bidentate moiety obtainedby removing two hydrogen atoms, one from each of two different aromaticring atoms of an aromatic compound, which moiety has from 6 to 10 ringatoms (unless otherwise specified). In an embodiment, each ring has from6 to 8 ring atoms. In this context, the prefix (e.g. C₆₋₁₀) denotes thenumber of ring atoms, or a range for the number of ring carbon atoms.

In some embodiments, substituents can themselves be substituted. Forexample, a C₁₋₁₂alkyl group may be substituted with, for example,hydroxy (referred to as a hydroxy-C₁₋₁₂alkyl group) or a halogen atom(referred to as a halo-C₁₋₁₂alkyl group), and a C₁₋₁₂alkoxy group may besubstituted with, for example, a halogen atom (referred to as ahalo-C₁₋₁₂alkoxy group).

The term “alkylene-arylene” used herein refers to a bidentate moietycomprising an alkylene moiety, -alkylene-, linked to an arylene moiety,-arylene-, that is, -alkylene-arylene-. Examples of alkylene-arylenegroups include C₁₋₂₀alkylene-C₆₋₁₀arylene, such as methylene-phenylene,ethylene-phenylene, propylene-phenylene, and ethenylene-phenylene (alsoknown as vinylene-phenylene).

The term “phosphinodithioate” as used herein refers to a >P(S)S⁻ group.

The term “phosphinodithioic acid” as used herein refers to a >P(S)SHgroup.

The term “protecting group” as used herein refers to a group capable ofprotecting a heteroatom (such as an oxygen atom), which protecting groupmay, subsequent to the reaction for which protection is employed, beremoved without disturbing the remainder of the molecule. Protectinggroups are well known and listed in standard texts such as Kocienski P.J., Protecting Groups, 3rd ed., Georg Thieme Verlag, New York, 2005; andGreene T. W., Wuts P. G. M., Protective Groups In Organic Synthesis, 3rded., John Wiley & Sons, New York, 1998.

Certain compounds may exist in one or more particular geometric,enantiomeric, diasteriomeric, tautomeric, or conformational forms.Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation andseparation of such isomeric forms are either known in the art.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, mean “including but not limited to”, anddo not exclude other moieties, additives, components, integers or steps.Moreover the singular encompasses the plural unless the contextotherwise requires: in particular, where the indefinite article is used,the specification is to be understood as contemplating plurality as wellas singularity, unless the context requires otherwise.

Preferred features of each aspect of the invention may be as describedin connection with any of the other aspects. Other features of theinvention will become apparent from the following examples. Generallyspeaking the invention extends to any novel one, or any novelcombination, of the features disclosed in this specification (includingany accompanying claims and drawings). Thus features, integers,characteristics, compounds, chemical moieties or groups described inconjunction with a particular aspect, embodiment or example of theinvention are to be understood to be applicable to any other aspect,embodiment or example described herein unless incompatible therewith.Moreover unless stated otherwise, any feature disclosed herein may bereplaced by an alternative feature serving the same or a similarpurpose.

Where upper and lower limits are quoted for a property, then a range ofvalues defined by a combination of any of the upper limits with any ofthe lower limits may also be implied.

In this specification, references to compound properties are—unlessstated otherwise—to properties measured under ambient conditions, i.e.at atmospheric pressure and at a temperature of from 16 to 22 or 25° C.,or from 18 to 22 or 25° C., for example about 20° C. or about 25° C.

The present invention will now be further described with reference tothe following non-limiting examples, and the accompanying illustrativedrawings, of which:

FIG. 1 shows percentage A431 cell death induced by photodynamic cellkilling following treatment with MAL in the absence and presence of theslow release hydrogen sulfide donor ADT-OH and its mitochondriallytargeted derivatives (AP39-C8, AP39-C10 and AP39-C12). ns=p>0.05,**=p<0.01, ***=p<0.001, Student's t-test compared to untreated control.+++=p<0.001, Student's t-test compared to the MAL group. Error barsrepresent one standard deviation, n=4.

FIG. 2 shows modes of A431 cell death induced by photodynamic cellkilling following treatment with MAL in the absence and presence of theslow release hydrogen sulfide donor ADT-OH and its mitochondriallytargeted derivatives (AP39-C8, AP39-C10 and AP39-C12). Error barsrepresent one standard deviation, n=4.

FIG. 3 shows percentage A431 cell death induced by photodynamic cellkilling following treatment with MAL in the absence and presence of theslow release hydrogen sulfide donor 4-HTB and its mitochondriallytargeted derivatives (AP123-C8, AP123-C10 and AP123-C12). ns=p>0.05,**=p<0.01, ***=p<0.001, Student's t-test compared to untreated control.++=p<0.01, +++=p<0.001, Student's t-test compared to the MAL group.Error bars represent one standard deviation, n=4.

FIG. 4 shows modes of A431 cell death induced by photodynamic cellkilling following treatment with MAL in the absence and presence of theslow release hydrogen sulfide donor 4-HTB and its mitochondriallytargeted derivatives (AP123-C8, AP123-C10 and AP123-C12). Error barsrepresent one standard deviation, n=4.

FIG. 5 shows the release of H₂S by AP39-C10 as measured by fluorogenicprobe WSP-1. The fluorescence of each well was measured withoutirradiation for 900 seconds, after which the wells were irradiated,which is the grey section in the figure. Following irradiation, a finalfluorescent measurement was recorded for each well (t=1200 s).***=p<0.001 compared to WSP-1+AP39. Representative of n=4.

FIG. 6 shows the release of H₂S by AP123-C10 as measured by fluorogenicprobe WSP-1. The fluorescence of each well was measured withoutirradiation for 900 seconds, after which the wells were irradiated,which is the grey section in the figure. Following irradiation, a finalfluorescent measurement was recorded for each well (t=1200 s).***=p<0.001 compared to WSP-1+AP123. Representative of n=4.

FIG. 7 shows a concentration-response plot of cell viability followingtreatment of A431 cells with the thioredoxin reductase inhibitor DNCB.Results are plotted as a percentage of viability compared to theuntreated control cells (0 μM DNCB). **=p<0.01, ***=p<0.001, Student'st-test compared to untreated cells. Error bars represent one standarddeviation, n=6.

FIG. 8 shows a concentration-response plot of cell viability followingtreatment of A431 cells with the thioredoxin reductase inhibitorauranofin. Results are plotted as a percentage of viability compared tothe untreated control cells (0 nM auranofin). Error bars represent onestandard deviation, n=5.

FIG. 9 shows cell death induced by photodynamic cell killing of A431cells treated with MAL in the absence and presence of the thioredoxinreductase inhibitors auranofin or DNCB. ***=p<0.001, Student's t-testcompared to untreated group. ++=p<0.01, +++=p<0.001, Student's t-testcompared to MAL-only group. Error bars represent one standard deviation,n=4.

FIG. 10 Modes of cell death following photodynamic cell killing of A431cells treated with MAL in the absence and presence of thioredoxinreductase inhibitors auranofin or DNCB. Error bars represent onestandard deviation, n=4.

FIG. 11 shows a concentration-response plot of cell viability followingtreatment of A431 cells with the superoxide scavengers TEMPO and TEMPOL.Results are plotted as a percentage of viability compared to theuntreated control cells (0 mM TEMPO or TEMPOL). *=p<0.05, **=p<0.01,***=p<0.001, Student's t-test compared to untreated cells. Error barsrepresent one standard deviation, n=4-5.

FIG. 12 shows cell death induced by photodynamic cell killing of A431cells treated with MAL in the absence and presence of TEMPO or TEMPOL.Error bars represent one standard deviation, n=6. **=p<0.01,***=p<0.001, Student's t-test compared to untreated group. +++=p<0.001,Student's t-test compared to MAL-only group.

FIG. 13 shows modes of cell death induced by photodynamic cell killingof A431 cells treated with MAL in the absence and presence of TEMPO orTEMPOL. Error bars represent one standard deviation, n=6.

FIG. 14 shows A431 PpIX accumulation following treatment with MAL in theabsence and presence of the small molecule antioxidants TEMPOL or TEMPO.Error bars represent one standard deviation, n=4. *=p<0.05, **=p<0.01,***=p<0.001, Student's t-test compared to 1 mM MAL-only.

FIG. 15 shows a concentration-response plot of cell viability followingtreatment of A431 cells with the mitochondria-targeted derivatives(AP39-C8, AP39-C10 and AP39-C12) of the non-targeted slow-releasinghydrogen sulfide donor, ADT-OH. Data are expressed as mean±S.Dpercentage of cell viability compared to untreated cells. n=4.

FIG. 16 shows a concentration-response plot of cell viability followingtreatment of A431 cells with the mitochondria-targeted derivatives(AP123-C8, AP123-C10 and AP123-C12) of the non-targeted slow-releasinghydrogen sulfide donor, 4-HTB. Data are expressed as mean±S.D percentageof cell viability compared to untreated cells. n=4.

FIG. 17 shows viability of A431 cells following treatment with AP39-C10and AP123-C10 for 72 hours. Data are expressed as mean±S.D. percentageof cell viability compared to untreated cells. *=p<0.05, ***=p<0.001,Student's t-test c.f. untreated cells (0 nM). n=6.

FIG. 18 shows A431 cell death induced by photodynamic cell killingfollowing treatment with MAL in the absence and presence of the slowrelease hydrogen sulfide donors AP39-C10 and AP123-C10. Data areexpressed as mean±S.D. percentage of cell death. ***=p<0.001, Student'st-test c.f. untreated control. +++=p<0.001, Student's t-test c.f. theMAL group. Error bars represent one standard deviation, n=5.

FIG. 19 shows modes of A431 cell death induced by photodynamic cellkilling following treatment with MAL in the absence and presence of theslow release hydrogen sulfide donors AP39-C10 and AP123-C10. Data areexpressed as mean±S.D. percentage cell death. n=5.

FIG. 20 shows PpIX accumulation in A431 cells following treatment withMAL in the absence and presence of non-targeted andmitochondria-targeted slow-releasing hydrogen sulfide donors. Data areexpressed as mean±S.D. arbitrary fluorescence units. ***=p<0.001,Student's t-test c.f. 1 mM MAL. n=4.

FIG. 21 shows the effects of AP39-C10 on MAL-induced PpIX accumulationin A431 cells. Data are expressed as mean±S.D. arbitrary fluorescenceunits. ***=p<0.001, Student's t-test c.f. 1 mM MAL. n=4.

FIG. 22 shows the effects of AP123-C10 on MAL-induced PpIX accumulationin A431 cells. Data are expressed as mean±S.D. arbitrary fluorescenceunits. ***=p<0.001, Student's t-test c.f. 1 mM MAL. n=4.

FIG. 23 shows the effects of AP39-C10 on reactive oxygen speciesgeneration during photodynamic irradiation of A431 cells pre-treatedwith MAL. Data are expressed as mean±S.D. percentage of untreated cells.***=p<0.001, Student's t-test c.f. the MAL group. n=4.

FIG. 24 shows the effects of AP123-C10 on reactive oxygen speciesgeneration during photodynamic irradiation of A431 cells pre-treatedwith MAL. Data are expressed as mean±S.D. percentage of untreated cells.***=p<0.001, Student's t-test c.f. the MAL group. n=4.

FIG. 25 shows a concentration-response plot of cell viability followingtreatment of A431 cells with inhibitors of the thioredoxin antioxidantsystem. Data are expressed as mean±S.D percentage of viability comparedto untreated cells. n=3.

FIG. 26 shows A431 cell death induced by photodynamic cell killingfollowing treatment with MAL in the absence and presence of thioredoxinantioxidant system inhibitors. Data are expressed mean±S.D. percentageof cell death. ***=p<0.001, Student's t-test c.f. untreated control.++=p<0.01, +++=p<0.001, Student's t-test c.f. MAL-PDT. Error barsrepresent one standard deviation, n=4.

FIG. 27 shows modes of A431 cell death induced by photodynamic cellkilling following treatment with MAL in the absence and presence ofthioredoxin reductase inhibitors and thioredoxin inhibitors. Data areexpressed mean±S.D. percentage of cell death. n=4.

FIG. 28 shows A431 cell death induced by photodynamic cell killingfollowing treatment with MAL in the absence and presence of differentconcentrations of auranofin. Data are expressed mean±S.D. percentage ofcell death. ***=p<0.001 Student's t-test c.f. untreated control.+++=p<0.001 Student's t-test c.f. MAL alone. n=4.

FIG. 29 shows modes of A431 cell death induced by photodynamic cellkilling following treatment with MAL in the absence and presence ofdifferent concentrations of auranofin. Data are expressed mean±S.D.percentage of cell death. n=4.

FIG. 30 shows PpIX accumulation in A431 cells following treatment withMAL in the absence and presence of thioredoxin antioxidant systeminhibitors. Data are expressed as mean±S.D. arbitrary fluorescenceunits. ***=p<0.001, Student's t-test c.f. MAL alone. n=4. a.u.=arbitraryunits.

FIG. 31 shows the effects of thioredoxin antioxidant system inhibitorson reactive oxygen species generation during photodynamic irradiation ofA431 cells pre-treated with MAL. Data are expressed mean±S.D. percentageof untreated cells. ***=p<0.001, Student's t-test c.f. untreatedcontrol, +++=p<0.001 c.f. MAL alone. n=5.

FIG. 32 shows a concentration-response plot of cell viability followingtreatment of A431 cells with the nitroxides TEMPO, TEMPOL, TEMPONE andMitoTEMPO. Data are expressed as mean±S.D percentage of viabilitycompared to untreated cells. n=3.

FIG. 33 shows A431 cell death induced by photodynamic cell killingfollowing treatment with MAL in the absence and presence of nitroxides.***=p<0.001, Student's t-test c.f. untreated control. ++=p<0.01,+++=p<0.001, Student's t-test c.f. MAL-PDT. Error bars represent onestandard deviation, n=4.

FIG. 34 legend shows modes of A431 cell death induced by photodynamiccell killing following treatment with MAL in the absence and presence ofnitroxides. Data are expressed mean±S.D. percentage of cell death. n=4.

FIG. 35 shows a concentration-response plot showing the effects of TEMPOand MitoTEMPO on MAL-induced PpIX accumulation in A431 cells. Data areexpressed mean±S.D. percentage increase in PpIX fluorescence compared toMAL alone. n=4.

FIG. 36 shows A431 cell death induced by photodynamic cell killingfollowing treatment with MAL in the absence and presence of differentconcentrations of TEMPOL. Data are expressed mean±S.D. percentage ofcell death. **=p<0.01, ***=p<0.001 Student's t-test c.f. untreatedcontrol. ++=p<0.01, +++=p<0.001 Student's t-test c.f. MAL alone. n=4.

FIG. 37 shows modes of A431 cell death induced by photodynamic cellkilling following treatment with MAL in the absence and presence ofnitroxides. Data are expressed mean±S.D. percentage of cell death. n=4.

FIG. 38 shows a concentration-response plot showing the effects ofTEMPOL, TEMPONE and TEMPO on MAL-induced PpIX accumulation in A431cells. Data are expressed mean±S.D. percentage change in PpIXfluorescence compared to MAL alone. **=p<0.01, ***=p<0.001 c.f. MALalone. n=4.

FIG. 39 shows the effects of nitroxides on reactive oxygen speciesgeneration during photodynamic irradiation of A431 cells pre-treatedwith MAL. Data are expressed mean±S.D. percentage of untreated cells.***=p<0.001, Student's t-test c.f. untreated control, +++=p<0.001 c.f.MAL alone. n=5.

FIG. 40 shows the effects of different concentrations of TEMPOL onreactive oxygen species generation during photodynamic irradiation ofA431 cells pre-treated with MAL. ***=p<0.001, Student's t-test c.f.untreated control, +=p<0.05, +++=p<0.001 c.f. MAL alone. Error barsrepresent one standard deviation, n=4.

EXPERIMENTAL METHODS

All solutions used in the following methods were pre-gassed with 2% O₂,5% CO₂ and 93% N₂, to investigate effects under a physiological [O₂] of2%.

Concentration-Response Toxicity Tests

A431 cells were seeded at a density of 1×10⁵ cells/ml in 96 well platesand incubated under 2% O₂ for 48 h prior to treatment. After 24 h, theculture medium was replaced and the cells were placed back into theincubators under 2% O₂. Following this incubation period, the culturemedium was then removed and the plates were washed with PBS. The cellswere then treated with 1 mM MAL in the absence or presence of one of thecompounds of interest and then incubated again at 2% O₂ for 3 h. Forexperiments carried out with SRHDs, this treatment step was carried outfor 5 h at 2% O₂. After treatment, the cells were washed with PBS, theculture medium was replaced and the cells were placed back under 2% O₂for 3 h. Following this final incubation, cell death was analysed usingthe resazurin viability assay.

Resazurin Microtitre Cell Viability Assay

Resazurin (7-hydroxy-3H-phenoxazin-3-one 0-oxide) is a weaklyfluorescent blue dye used primarily in oxidation-reduction cellviability assays. Irreversible NADH-dependent reduction by themitochondria in mammalian cells leads to the production of resorufin, ahighly fluorescent product with excitation at 571 nm, emission at 585nm. As the reduction of resazurin to resorufin is primarily driven bymitochondria it can be used as an estimate of cell viability wheninvestigating the toxicity of compounds, as dead or dying cells have adecreased mitochondrial activity, producing less resorufin and thereforeless fluorescence. Using resazurin in conjunction with microtitre (96well) plates allows high throughput screening of compounds, which canlater be confirmed by other, more definitive methods such as flowcytometry.

A431 cells were seeded into 96 well plates at a density of 1.5*10⁶ cellsper ml (200 μl per well, 3*10⁵ cells) and incubated for 24 h to adhere.Following incubation, the cells were washed and treated with thecompound of interest for the relevant time, and then washed with PBS. 55μM resazurin was diluted to 10% (5.5 μM) in fresh culture medium andapplied to the cells. After a 2 h incubation, the fluorescence of eachwell was measured (excitation: 571 nm, emission: 585 nm) using afluorescence plate reader. Data from these experiments was plotted as apercentage of cell viability compared to untreated control cells.

Photodynamic Cell Killing in the Presence of Slow Release HydrogenSulfide Donors (SRHDs)

A431 cells (human epithelial squamous cell carcinoma cells) were seededat a density of 1×10⁶ cells/ml in T12.5 cm² flasks and incubated under2% O₂ for 48 h prior to treatment. After 24 h, the culture medium wasreplaced and the cells were placed back into the incubators under 2% O₂.Following this incubation period, the culture medium was then removedand the flasks were washed with PBS. The cells were then treated withone of the SRHDs and incubated at 2% O₂ for 2 h, after which MAL wasadded to a final concentration of 1 mM and the cells were incubated at2% O₂ for 3 h. This meant in total, the cells were treated with theSRHDs for 5 h.

After this treatment, the cells were irradiated for 5 min (630 nm, 25J/cm²), then washed with PBS and the culture medium was replaced, afterwhich the cells were placed back under 2% O₂ and incubated for a further3 h. Following this final incubation, cell death was analysed by annexinV-FITC and propidium iodide staining in conjunction with flow cytometry.

Photodynamic Cell Killing in the Presence of Inhibitors of theThioredoxin Antioxidant System or Nitroxides

A431 cells (human epithelial squamous cell carcinoma cells) were seededat a density of 1×10⁶ cells/ml in T12.5 cm² flasks and incubated under2% O₂ for 48 h prior to treatment. After 24 h, the culture medium wasreplaced and the cells were placed back into the incubators under 2% O₂.Following this incubation period, the culture medium was then removedand the flasks were washed with PBS. The cells were then treated with 1mM MAL in the absence or presence of one of the chosen inhibitors of thethioredoxin antioxidant system, or nitroxides, and then incubated againat 2% O₂ for 3 h.

After this treatment, the cells were irradiated for 5 min (630 nm, 25J/cm²), then washed with PBS and the culture medium was replaced, afterwhich the cells were placed back under 2% O₂ and incubated for a further3 h. Following this final incubation, cell death was analysed by annexinV-FITC and propidium iodide staining in conjunction with flow cytometry.

Flow Cytometry of Annexin V-FITC and Propidium Iodide Stained Cells

Cell death analysis by flow cytometry was carried out using the annexinV-FITC/propidium iodide protocol. This protocol allows a quantitativeassessment of cell death, particularly specific modes of death, andinvolves staining the cells with fluorescein isothiocyanate-conjugatedannexin A5 (annexin V-FITC) and propidium iodide (PI). Positive stainingwith annexin V-FITC alone indicates an apoptotic cell; positive stainingwith PI alone indicates a necrotic cell and dual staining of bothannexin V and PI indicates a “late apoptotic” cell.

From each flask being assessed, the old medium was removed and placedinto a corresponding individual 15 ml Falcon tube in order to collectany cells that may have detached from the surface. The flask was washedonce with 1 ml PBS; also deposited in the Falcon tubes. Trypsin (500 μl)was added to each flask and the flasks were returned to the incubatorwith their caps loosened. Cell detachment was monitored using aninverted light microscope, until 50% of the cells were detached. Theflasks were then lightly tapped to detach the remainder of the cells.Medium (2 ml) was added to each flask in order to neutralise the trypsinpresent before being removed and dispensed into the Falcon tubes. Onemore wash with PBS was carried out in order to ensure any cells left inthe flasks were collected. The Falcon tubes were then centrifuged at 490g for 3 min, forming a pellet at the bottom of the tube. The supernatantwas discarded, and the cells were re-suspended in 5 ml of PBS, in orderto wash them, before being centrifuged again at 490 g for 3 min. Thewash stage was carried out once more before re-suspending the cellpellet in 95 μl of ice cold calcium (Ca²⁺) buffer (50 mM HEPES, 700 mMNaCl, 12.5 mM CaCl2, pH 7.4) and adding 5 μl 12.5 μg/ml of annexinV-FITC (final concentration, 1.25 μg/ml), under reduced light conditionsto prevent bleaching of the FITC. The Falcon tubes were placed on iceand in the dark for 15 min to allow annexin V staining. After 15 min,860 μl of Ca²⁺ buffer (10 mM Hepes adjusted to pH 7.4, 140 mM NaCl and2.5 mM CaCl₂) was added, followed by 40 μl of 1 mg/ml PI in water (finalconcentration, 0.04 mg/ml) to give a final volume of 1 ml. The sampleswere then ready to be assessed by flow cytometry.

The following flow cytometry process of sample detection was used. Whenusing annexin V-FITC and PI, the detectors being used were FL1(λ_(max)=520 nm) and FL3 (λ_(max)=670 nm), respectively, each with theirown logarithmic histogram generated by the software. The intensity ofthe fluorescence detected is measured on a logarithmic scale on thex-axis and the y-axis represents the number of cells detected at a givenfluorescence intensity. A plot of FL1/FL3 produces a 4-quadrant graph,where unstained and untreated cells are located in the bottom leftquadrant, representing any cells found in the 1^(st) decade of thesingle plot logarithmic histograms. Single staining with annexin V-FITCand PI are located in the bottom right and top left quadrant,respectively, and dual staining is located in the top right quadrant.These quadrants represent any cells detected above the 1^(st) decade ofthe logarithmic histograms.

Effects of Irradiation of PpIX in the Presence of Slow Release HydrogenSulfide Donors (SRHDs) in a Cell Free System

Fluorogenic probe WSP-1(3′-methoxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-6′-yl2-(pyridin-2-yldisulfanyl)benzoate) can be used to detect H₂S. WSP-1reacts with H₂S to form a fluorescent product (Liu, C. et al., AngewChem. Int. Ed. Engl. 2011, 50, 10327-10329; Cortese-Krott, M. M. et al.,Redox Biol. 2014, 2, 234-244.)

In a cell free system, solutions of the H₂S-detecting fluorogenic probeWSP-1 (100 μM) were made up in the absence and presence of AP39-C10 andAP123-C10 (100 μM) and in the absence and presence of PpIX (2 μM). Forthose solutions containing AP39-C10, the reducing agent DTT (100 μM) wasalso added as AP39-C10 does not release H₂S in the absence of areductant. The solution was pipetted into individual wells of a 96 wellplate and the fluorescence of each well was measured (excitation 465 nm,emission 515 nm) using a SpectraMax M2e fluorescent plate reader, every20 seconds for a period of 15 minutes (900 s). After this was completed,the wells were irradiated (636±5 nm, 25 J/cm²). Following irradiation, afinal fluorescent measurement was recorded for each well (t=1200 s).

Effects of Nitroxides on PpIX Accumulation

A431 cells were seeded into 96 well plates at a density of 1.5*10⁶ cellsper ml (200 μl per well, 3*10⁵ cells) and incubated for 24 h to adhere.The cells were then treated with 1 mM MAL in the absence or presence ofa range of concentrations of TEMPOL or TEMPO and incubated again at 2%O₂ for 3 h. After treatment, the cells were washed with PBS and a final100 μl PBS was pipetted into each well for fluorescence readings. Thefluorescence of PpIX was measured using a BMG Pherastar plate readerwith a 410 nm excitation filter and a 630 nm emission filter.

EXAMPLE 1 Effects of Slow Release Hydrogen Sulfide Donors onPhotodynamic Cell Killing

Compounds Tested

The effects of slow-releasing H₂S donors (SRHDs) on photodynamic cellkilling were investigated by using mitochondrially targeted compoundscontaining a triphenylphosphonium cation (TPP⁺) as the mitochondrialtargeting group with two different H₂S releasing moieties and differingchain lengths.

“AP39” compounds AP39-C8, AP39-C10 and AP39-C12 are relatedmitochondrially targeted slow-releasing H₂S donors with differing chainlengths, which have the following structures:

“AP123” compounds AP123-C8, AP123-C10 and AP123-C12 are relatedmitochondrially targeted slow-releasing H₂S donors with differing chainlengths, which have the following structures:

The AP39 and AP123 compounds can be prepared using the method describedin WO 2013/045951.

Additionally, the H₂S releasing moieties of the AP39 and AP123compounds, 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH) and4-hydroxythiobenzamide (4-HTB) were also used for comparison.

ADT-OH can be prepared using the method described in US 2008/0004245.

4-HTB is commercially available from Sigma-Aldrich.

Slow-releasing H₂S donor GYY4137 was also used for comparison. GYY4137is a non-targeted slow-releasing H₂S donor of the following structure:

GYY4137 is commercially available or can be prepared using the methoddescribed in Li L et al., Circulation 2008, 117:2351-2360.

Results

A431 cells were treated with 1 mM MAL in the absence and presence of anSRHD (1 μM) and with or without irradiation. Cell viability was assessed3 hours post-irradiation/post-treatment. For the cells treated with MALin the presence of an SRHD, in order to allow for H₂S to accumulate,incubation with the SRHD was carried out for a total of 5 hours. Thecells were treated with the SRHDs 2 hours prior to addition of MAL,followed by incubation with MAL for 3 h. The cells were then irradiatedand cell viability was measured 3 hours post-irradiation.

Measurements of total cell death following treatment of the cells withADT-OH and its AP39 derivatives have been plotted in FIG. 1, and abreakdown of the cell death types has been plotted in FIG. 2.Measurements of total cell death following treatment of the cells with4-HTB and its AP123 derivatives have been plotted in FIG. 3, and abreakdown of the cell death types has been plotted in FIG. 4. The datafor these experiments were collected at the same time but have beensplit up in order to present the results for ADT-OH and its AP39derivatives and for 4-HTB and its AP123 derivatives in a clear way. Asthese experiments were carried out at the same time, data for theuntreated controls, MAL, and GYY4137 is the same in FIGS. 1 and 3, andin FIGS. 2 and 4.

As can be seen from FIGS. 1 and 3, the mean cell death of the untreatedcontrol group was 10.1±3.4%.

Dark toxicity tests (without irradiation) of the ADT-OH derivativesfound that these compounds did not induce any statistically significantchanges in cell viability compared with the untreated control group.Cell death was found to be 14.9±2.3%, 12.1±4.9% and 11.7±1.5% forAP39-C8, AP39-C10 and AP39-C12, respectively (FIG. 1).

Dark toxicity tests (without irradiation) of the 4-HTB derivatives alsofound no statistically significant changes in cell viability comparedwith the untreated control group. Cell death was found to be 13.2±3.1%,14.5±0.5% and 12.7±3.0% for AP123-C8, AP123-C10 and AP123-C12,respectively (see FIG. 3).

None of the SRHDs were toxic in the absence of irradiation, suggestingthat they were well tolerated by A431 cells during the 5 h incubation.

Treatment with 1 mM MAL and irradiation in the absence of any SRHDresulted in 27.5±5.5% cell death (p<0.01 compared to untreated control)(see FIGS. 1 and 3).

Co-treatment with 1 mM MAL and irradiation in the presence of ADT-OHinduced 25.7±6% (p>0.05 compared to MAL and irradiation) cell death (seeFIG. 1).

Co-treatment with 1 mM MAL and irradiation in the presence of 4-HTBresulted in 22.5±5.5% (p>0.05 compared to MAL and irradiation) celldeath (see FIG. 3).

A co-treatment of 1 mM MAL with GYY4137 did not significantly alter cellviability (27.1±6.0%, p>0.05) compared to treatment with MAL andirradiation (see FIGS. 1 and 3).

Co-treatment with 1 mM MAL and irradiation in the presence of the AP39compounds induced 46.3±2.1% (p<0.001), 45.3±1.9% (p<0.001) and 51.6±3.2%(p<0.001) cell death for AP39-C8, AP39-C10 and AP39-C12, respectively.

Co-treatment with 1 mM MAL and irradiation in the presence of the AP123compounds resulted in 67.5±6.4% (p<0.001), 50.9±6.5% (p<0.01) and72.5±2.0% (p<0.001) cell death for AP123-C8, AP123-C10 and AP123-C12,respectively.

Treatment with MAL and irradiation in the absence of the SRHDssignificantly increased cell death compared to untreated controls(p<0.01). The non-targeted H₂S releasing moieties of AP39 (ADT-OH) andAP123 (4-HTB) had no statistically significant effect on photodynamiccell killing, exhibiting similar levels of cell death compared to MALand irradiation only (p>0.05). GYY4137 was also found to have no effecton photodynamic cell killing (p>0.05). All derivatives of AP39 andAP123, however, significantly increased cell killing compared to MALonly with irradiation (p<0.001).

As mentioned above, FIGS. 2 and 4 show a breakdown of the cell deathtypes. Annexin V-FITC and propidium iodide staining was used to assessthe modes of cell death.

Treatment with 1 mM MAL and irradiation resulted in an increase in alltypes of cell death compared to untreated controls. Co-treatment withthe AP39 derivatives primarily increased total cell death throughincreased early apoptosis, with smaller increases in late apoptosis.Necrotic cell death in the presence of AP39 derivatives appeared todecrease (see FIG. 2). The C8 and C10 AP123 derivatives increased totalcell death through increases in early and late apoptosis, with littleeffect on necrosis. AP123-C12 had less effect on early apoptosis,instead increasing late apoptosis and necrosis (see FIG. 4). ADT-OH,4-HTB and GYY4137 had no effect on the types of cell death observedcompared to treatment with MAL and irradiation, with each treatmentresulting in similar levels of early and late apoptosis and necrosis.

Further experiments were carried out to investigate the effects ofirradiation on the release of H₂S by H₂S donors AP39-C10 and AP123-C10in the absence and presence of PpIX. Experiments were carried out in acell-free system and monitored by using the H₂S-sensitive fluorogenicprobe WSP-1. The results of are set out in FIGS. 5 and 6.

As can be seen from FIGS. 5 and 6, prior to irradiation, H₂S release bythe C10 variants of AP39 and AP123 appears to be unaffected by thepresence of PpIX. Irradiation of the donors in the absence of PpIX alsodoes not have any significant effect on the release of H₂S. In thepresence of PpIX, however, irradiation significantly increases therelease of H₂S by both AP39 and AP123 (p<0.001).

FIGS. 5 and 6 also show that the ADT-OH derivative AP123-C10 releasesmore H₂S than the 4-HTB derivative AP39-C10.

EXAMPLE 1A Mitochondria-Targeted Slow Release Hydrogen Sulfide Donors(SRHDs) Potentiate Methyl Aminolaevulinic Acid-Based Photodynamic CellKilling

The data covered in this Example build on the previously identifiedabilities of mitochondria-targeted hydrogen sulfide donors to potentiatemethyl aminolaevulinic acid (MAL; Metvix®) photodynamic cell killing.

In short, these data show that the mitochondria-targeted derivatives ofADT-OH (AP39-C8, AP39-C10 and AP39-C12) and 4-HTB (AP123-C8, AP123-C10and AP123-C12) are well tolerated across a large range of concentrationsand only begin to exhibit cytotoxicity well outside of the “therapeutic”concentration. This tolerance is exhibited over periods relevant to thephotodynamic experiments carried out (5 hours) and over prolongedperiods of exposure (72 hours), suggesting the compounds are safe.

AP39-C10 and AP123-C10 exhibit a potentiating effect at concentrationsas low as 10 nM, where a ˜2-fold increase in cell killing is observedcompared to the use of MAL alone. This is significantly more potent(5000-fold) than CP94 (50 μM) which exerts a similar degree ofpotentiation. The observed increases in total cell death were driven byincreases in early and late apoptosis, with little-to-no effect onnecrosis.

By measuring the effects of these compounds on MAL-inducedprotoporphyrin IX (PpIX) accumulation, it was possible to establish thatthese compounds do not exert their effects through the “traditional”method of increasing PpIX accumulation (which is how CP94 exerts itseffects).

A small (but statistically significant) increase in mitochondrialoxidant generation was detected following photodynamic irradiation ofcells co-treated with MAL and AP39-C10 or AP123-C10. This increase inoxidant generation appears to occur in a concentration-dependent manner.

FIG. 15—Dark Toxicity of Non-Targeted Slow Releasing Hydrogen SulfideDonor ADT-OH and its Mitochondria-Targeted Derivatives, as Measured bythe Viability of Treated A431 Cells

A431 cells were treated with the non-targeted slow releasing hydrogensulfide donor ADT-OH and its mitochondria-targeted derivatives (AP39-C8,AP39-C10 and AP39-C12) at a range of concentrations of each compound(0.1-500 μM), for 5 hours. Following this treatment, viability wasmeasured using a resazurin-based fluorescence assay. The mean viabilityof the cells following treatment was calculated as a percentage of cellviability compared to the untreated controls.

The data in FIG. 15 show that treatment with each compound was welltolerated up to a concentration of 100 μM. At 500 μM, all compoundsexhibited statistically significant cytotoxicity, with ADT-OH, AP39-C8,AP39-C10 and AP39-C12 decreasing viability to 76.7±1.3% (p<0.01),20.1±4.5% (p<0.001), 59.3±13.3% (p<0.05) and 13.3±1.8% of untreatedcontrol (p<0.01), respectively.

FIG. 16—Dark Toxicity of Non-Targeted Slow Releasing Hydrogen SulfideDonor 4-HTB and its Mitochondria-Targeted Derivatives, as Measured bythe Viability of Treated A431 Cells

A431 cells were treated with the non-targeted slow releasing hydrogensulfide donor 4-HTB and its mitochondria-targeted derivatives (AP123-C8,AP123-C10 and AP123-C12) at a range of concentrations of each compound(0.1-500 μM), for 5 hours. Following this treatment, viability wasmeasured using a resazurin-based fluorescence assay. The mean viabilityof the cells following treatment was calculated as a percentage of cellviability compared to the untreated controls.

The data in FIG. 16 show that treatment with each compound was welltolerated up to a concentration of 100 μM. 4-HTB was also tolerated upto 500 μM, whilst AP123-C8, AP123-C10 and AP123-C12 exhibitedstatistically significant cytotoxicity at this concentration, decreasingviability to 13.2±1.8% (p<0.001), 62.6±9.1% (p<0.05) and 17.0±0.7% ofuntreated control (p<0.001), respectively.

FIG. 17—Viability of A431 Cells Following Treatment with AP39-C10 andAP123-C10 for 72 Hours: AP39-C10 and AP123-C10 Did not InduceSignificant Cytotoxicity in A431 Cells Following a 72 Hour Treatmentwith Selected “Therapeutic” Concentrations

To ensure that these compounds were not toxic at selected “therapeutic”concentrations during prolonged treatment, A431 cells were treated witha range of concentrations (0-1000 nM) of each of AP39-C10 and AP123-C10for 72 hours, after which viability was assessed using a resazurin-basedfluorescence assay. A positive control, where cells were treated with 30μM etoposide for 24 hours was also carried out.

The mean viability of the cells following treatment was calculated as apercentage of viability compared to the untreated controls (0nM—AP39-C10; 100.0±2.6%, AP123-C10; 100.0±5.4%).

The data in FIG. 17 show that, over 72 hours, treatment with AP39-C10and AP123-C10 was well tolerated at each concentration tested. Whentreated with 1000 nM AP39-C10, cell exhibited a small, but statisticallysignificant, decrease in viability (94.1±2.6%, p<0.05 c.f. control). Apositive control, where cells were treated with the chemotherapeuticagent etoposide (30 μM), significantly decreased cell viability (p<0.001c.f. control).

FIG. 18—A431 Cell Death Induced by Photodynamic Cell Killing FollowingTreatment with MAL in the Absence and Presence of the Slow ReleaseHydrogen Sulfide Donors AP39-C10 and AP123-C10: AP39-C10 and AP123-C10Significantly Increase MAL-Based Photodynamic Cell Killing

A431 cells were treated concurrently with AP39-C10 or AP123-C10 (10, 100or 1000 nM) for 5 hours and MAL (1 mM) for 3 hours, after which cellswere irradiated for 5 minutes (630 nm, 25 J/cm²) and then incubated forfurther 3 hours. Cell viability was then assessed by annexin V-FITC andpropidium iodide staining in conjunction with flow cytometry.Co-treatment with CP94 (50 μM) (a clinically used iron chelator) wasused as a positive control and for comparison. Results are set out inFIG. 18.

The mean cell death of the untreated control group was 13.8±1.6%.Treatment with 1 mM MAL in the absence of any SRHD resulted in asignificant increase in cell death (32.8±2.5%, p<0.001 c.f. control).Carrying out photodynamic treatment with 1 mM MAL in the presence of 10,100 or 1000 nM of AP39-C10 resulted in further increases in cell death,with 50.7±3.1% (p<0.001 c.f. MAL), 53.1±2.3% (p<0.001) and 50.2±4.9%(p<0.001) cell death, respectively.

Photodynamic treatment with 1 mM MAL in the presence of 10, 100 or 1000nM of AP123-C10 also resulted in further increases in cell death, with57.2±1.3% (p<0.001 c.f. MAL alone), 56.5±2.7% (p<0.001 c.f. MAL alone)and 66.7±5.6% (p<0.001 c.f. MAL alone) cell death, respectively.

FIG. 19—Modes of A431 Cell Death Induced by Photodynamic Cell KillingFollowing Treatment with MAL in the Absence and Presence of the SlowRelease Hydrogen Sulfide Donors AP39-C10 and AP123-C10: AP39-C10 andAP123-C10 Significantly Increase MAL-Based Photodynamic Cell KillingThrough Selective Promotion of Apoptotic Cell Death

Annexin V-FITC and propidium iodide staining was used to assess themodes of cell death following irradiation of A431 cells treated with 1mM MAL in absence and presence of AP39-C10 or AP123-C10 (10, 100 or 1000nM). Co-treatment with CP94 (50 μM) was used as a positive control andfor comparison. Results are set out in FIG. 19.

Photodynamic irradiation of A431 cells pre-treated with 1 mM MALresulted in an increase in all types of cell death compared to theuntreated controls. Co-treatment with AP39-C10 or AP123-C10 at 10, 100or 1000 nM resulted in a further increase in apoptosis and lateapoptosis, with no apparent effect on necrotic cell death.

FIG. 20—PpIX Accumulation in A431 Cells Following Treatment with MAL inthe Absence and Presence of Non-Targeted and Mitochondria-TargetedSlow-Releasing Hydrogen Sulfide Donors: Slow Releasing Hydrogen SulfideDonors Did not Increase MAL-Induced Protoporphyrin IX Accumulation

A431 cells were treated concurrently with slow-releasing hydrogensulfide donors (1 μM) for 5 hours and MAL (1 mM) for 3 hours, afterwhich PpIX accumulation was measured by fluorescence plate reader(excitation 410 nm, excitation 630 nm). Co-treatment with CP94 (50 μM)was used as a positive control and for comparison.

The effects of ADT-OH, 4-HTB and their mitochondria-targeted derivatives(1 μM) on PpIX accumulation were investigated. The data presented inFIG. 20 show the measured relative fluorescence of PpIX. 1 mM MALsignificantly increased PpIX accumulation compared to untreated controls(p<0.001). In sharp contrast to CP94 (an iron chelator and well-knownenhancer of PpIX accumulation used clinically), none of theslow-releasing hydrogen sulfide donors exhibited any effects on PpIXaccumulation (p>0.05 c.f. 1 mM MAL) e.g. inhibition or furtheraccumulation. CP94 significantly increased PpIX accumulation compared to1 mM MAL alone (p<0.001).

FIG. 21—the Effects of AP39-C10 on MAL-Induced PpIX Accumulation in A431Cells: AP39-C10 had No Effect on MAL-Induced Protoporphyrin IXAccumulation

A431 cells were treated concurrently with AP39-C10 (10, 100 or 1000 nM)for 5 hours and MAL (1 mM) for 3 hours, after which PpIX accumulationwas measured by fluorescence plate reader (excitation 410 nm, excitation630 nm). Co-treatment with CP94 (50 μM) was used as a positive controland for comparison.

The effects of AP39-C10 (10, 100 and 1000 nM) on PpIX accumulation wereinvestigated. The data presented in FIG. 21 show the measured relativefluorescence of PpIX. 1 mM MAL significantly increased PpIX accumulationcompared to untreated controls (p<0.001). AP39-C10 had no effect on PpIXaccumulation across any of the tested concentrations (p>0.05 c.f. 1 mMMAL). A positive control, the iron chelator CP94, significantlyincreased PpIX accumulation compared to 1 mM MAL alone (p<0.001).

FIG. 22—the Effects of AP123-C10 on MAL-Induced PpIX Accumulation inA431 Cells: AP123-C10 had No Effect on MAL-Induced Protoporphyrin IXAccumulation

A431 cells were treated concurrently with AP123-C10 (10, 100 or 1000 nM)for 5 hours and MAL (1 mM) for 3 hours, after which PpIX accumulationwas measured by fluorescence plate reader (excitation 410 nm, excitation630 nm). Co-treatment with CP94 (50 μM) was used as a positive controland for comparison.

The effects of AP123-C10 (10, 100 and 1000 nM) on PpIX accumulation wereinvestigated. The data presented in FIG. 22 show the measured relativefluorescence of PpIX. 1 mM MAL significantly increased PpIX accumulationcompared to untreated controls (p<0.001). AP123-C10 had no effect onPpIX accumulation across any of the tested concentrations (p>0.05 c.f. 1mM MAL). A positive control, the iron chelator CP94, significantlyincreased PpIX accumulation compared to 1 mM MAL alone (p<0.001).

FIG. 23—the Effects of AP39-C10 on Reactive Oxygen Species GenerationDuring Photodynamic Irradiation of A431 Cells Pre-Treated with MAL:AP39-C10 Increases Mitochondrial Oxidant Generation During MAL-BasedPhotodynamic Cell Killing

A431 cells were treated concurrently with AP39-C10 (10, 100 or 1000 nM)for 5 hours, MAL (1 mM) for 3 hours and MitoSOX (2.5 μM) for 1 hour,after which they were irradiated for 5 min (630 nm, 25 J/cm²) andMitoSOX (mito-2-OH-E⁺) fluorescence, indicative of mitochondrial oxidantproduction, was immediately analysed by flow cytometry. Results are setout in FIG. 23.

Mitochondria-targeted dihydroethidium (i.e. MitoSOX) was used to assessthe production of mitochondrial reactive oxygen species (ROS) by PpIXphotochemical reactions and to determine the effects of AP39-C10 on thisprocess. All of the results are represented as a percentage of theuntreated controls (100.0±8.5%). The fluorescence of the photoirradiatedcells, which had been pre-treated with MAL, was 193.7±7.3% (p<0.001 c.f.untreated controls). Co-treatment of MAL with 10 nM AP39-C10 resulted ina small, statistically non-significant, increase in this fluorescence to207.3±10.7% (p>0.05 c.f. MAL). Co-treatment of MAL with 100 or 1000 nMAP39-C10 resulted in significant increases to 227.1±3.5% (p<0.001) and214.3±6.4% (p<0.001).

FIG. 24—the Effects of AP123-C10 on Reactive Oxygen Species GenerationDuring Photodynamic Irradiation of A431 Cells Pre-Treated with MAL:AP123-C10 Increases Mitochondrial Oxidant Generation During MAL-BasedPhotodynamic Cell Killing

A431 cells were treated concurrently with AP123-C10 (10, 100 or 1000 nM)for 5 hours, MAL (1 mM) for 3 hours and MitoSOX (2.5 μM) for 1 hour,after which they were irradiated for 5 min (630 nm, 25 J/cm²) andMitoSOX (mito-2-OH-E⁺) fluorescence, indicative of mitochondrial oxidantproduction, was immediately analysed by flow cytometry. Results are setout in FIG. 24.

Mitochondria-targeted dihydroethidium was used to assess the productionof mitochondrial reactive oxygen species (ROS) by PpIX photochemicalreactions and to determine the effects of AP123-C10 on this process. Allof the results are represented as a percentage of the untreated controls(100.0±5.4%). The fluorescence of the photoirradiated cells, which hadbeen pre-treated with MAL, was 220.1±8.5% (p<0.001 c.f. untreatedcontrols). Co-treatment of MAL with 10, 100 or 1000 nM AP123-C10resulted in significant increases in this fluorescence to 237.4±8.2%(p<0.01 c.f. MAL), 245.3±10.6% (p<0.01) and 248.1±15.1% (p<0.01),respectively.

EXAMPLE 2 Effects of Thioredoxin Reductase Inhibitors on PhotodynamicCell Killing

Compounds Tested

The effects of thioredoxin reductase inhibitors on photodynamic cellkilling were investigated by using the thioredoxin reductase inhibitorsauranofin and 2,4-dinitrochlorobenzene (DNCB), which have the followingstructures:

Auranofin is commercially available from Sigma Aldrich and Enzo LifeSciences.

DNCB is commercially available from Sigma Aldrich.

Toxicity of Thioredoxin Reductase Inhibitors Auranofin and2,4-Dinitrochlorobenzene (DNCB)

Initial experiments were carried out to establish the toxicity ofauranofin and DNCB under 2% O₂ as measured using a resazurin oxidationassay, to provide an estimation of viability based on cellular metabolicactivity.

A431 cells were treated with 1 mM MAL±DNCB (0-50 μM) for 3 hours, afterwhich cell viability was measured using a resazurin-based fluorescenceassay (FIG. 7). The mean viability of cells following treatment wascalculated as a percentage of viability compared to the untreatedcontrol cells (100.0±11.1%). Treatment with DNCB at concentrations of 1,5 and 10 μM did not significantly affect cell viability, with 97.4±6.9%,99.4±7.3% and 98.6±6.2% viability, respectively. In the presence of DNCBat a concentration of 25 μM cell viability statistically significantlydecreased to 79.0±4.3% (p<0.01) and 50 μM DNCB decreased viabilityfurther, to 57.7±1.4% (p<0.001).

A431 cells were treated with 1 mM MAL±auranofin (0-2000 nM) for 3 hours,after which cell viability was measured using a resazurin-basedfluorescence assay (FIG. 8). The mean viability of cells followingtreatment was calculated as a percentage of viability compared to theuntreated control cells (100.0±8.2%). Cells treated with 100, 250, 500,1000 and 2000 nM auranofin exhibited 95.8±3.6%, 96.7±7.8%, 96.2±3.7%,100.0±6.1% and 100.1±5.9% viability, respectively. None of these resultswere statistically significantly different compared to untreated controlcells.

From these experiments, concentrations of 100 nM auranofin and 10 μMDNCB were chosen for further experimentation.

The Effects of Co-Treatment with Thioredoxin Reductase Inhibitors DNCBor Auranofin on MAL-Based Photodynamic Cell Killing

A431 cells were treated with 1 mM MAL in absence and presence ofauranofin (100 nM) or DNCB (10 μM) for 3 h, after which cells wereirradiated for 5 min (630 nm, 25 J/cm²) and then incubated for further 3h, so A431 cell viability was assessed 3 hours post-irradiation.Viability was assessed by annexin V-FITC and propidium iodide stainingin conjunction with flow cytometry.

Measurements of total cell death following treatment of the cells withMAL in the absence and presence of the thioredoxin reductase inhibitorsauranofin or DNCB have been plotted in FIG. 9. The mean cell death ofthe untreated control group was 9.0±2.0%.

Treatment with 1 mM MAL and irradiation in the absence of co-treatmentresulted in 32.5±3.6% cell death; a statistically significant increasecompared to untreated controls (p<0.001).

Co-treatment with 1 mM MAL, 10 μM DNCB or 100 nM auranofin andirradiation resulted in 46.3±4.0% and 65.5±1.7% cell death,respectively. In the presence of 10 μM DNCB, photodynamic cell killingwas significantly increased compared to MAL and irradiation only(p<0.01). In the presence of 100 nM auranofin, cell killing was alsosignificantly increased compared to MAL and irradiation only (p<0.001).Auranofin also increased cell killing more than DNCB (p<0.001),indicating that its sensitising effects are considerably more potentthan DNCB as a greater effect was observed using a concentration 100times lower.

A breakdown of A431 cell death for each treatment has been plotted inFIG. 10. Annexin V-FITC and propidium iodide staining was used to assessthe modes of cell death.

Treatment with 1 mM MAL and irradiation resulted in an increase in alltypes of cell death compared to untreated controls.

Co-treatment with either DNCB or auranofin followed by irradiationprimarily increased necrotic cell death (propidium iodide staining)compared to 1 mM MAL and irradiation. Co-treatment with auranofin alsoslightly increased late apoptotic cell death compared to 1 mM MAL andirradiation.

EXAMPLE 2A Thioredoxin Reductase Inhibitors and Thioredoxin InhibitorsPotentiate Methyl-Aminolaevulinic Acid-Based Photodynamic Cell Killing

The data covered in this Example build on the previously identifiedabilities of inhibitors of the thioredoxin antioxidant system topotentiate methyl-aminolaevulinic acid (MAL; Metvix®) photodynamic cellkilling.

This work includes the use of compounds known as gold (I) thiolates,which covers auranofin (AUR), aurothiomalate (ATM) and aurothioglucose(ATG) as well as an alkylating agent, dinitrochlorobenzene (DNCB). These4 compounds are known to inhibit thioredoxin reductase. Additionally,the thioredoxin inhibitor PX12 (2-[(1-Methylpropyl)dithio]-1H-imidazole)has been investigated.

These data show that each of the inhibitors are well tolerated across alarge range of concentrations and only begin to exhibit cytotoxicityoutside of the “therapeutic” concentration. This tolerance is exhibitedover periods relevant to the photodynamic experiments carried out (24hours), suggesting the compounds are safe at the concentrations carriedforward into further investigation.

DNCB exhibits a mild potentiating effect, whilst AUR, ATM, ATG and PX12exhibit a more robust potentiating effect. The observed increases intotal cell death were driven by increases in late apoptosis andnecrosis, with little-to-no effect on early apoptosis. Furtherinvestigation revealed that the potentiating effects of AUR areconcentration-dependent and a concentration of 100 nM exerted a similardegree of potentiation as CP94 (50 μM), making AUR 500-fold more potent.

By measuring the effects of these compounds on MAL-inducedprotoporphyrin IX (PpIX) accumulation, it was possible to establish thatnone of the inhibitors of the thioredoxin antioxidant system exert theireffects through the “traditional” method of increasing PpIX accumulation(which is how CP94 exerts its effects).

A significant increase in mitochondrial oxidant generation was detectedfollowing photodynamic irradiation of cells co-treated with MAL and eachof the inhibitors, providing a mechanism by which these potentiatingeffects are exerted.

-   Key:-   AUR=auranofin-   ATM=aurothiomalate-   ATG=aurothioglucose-   DNCB=dichloronitrobenzene    FIG. 25—Dark Toxicity of Thioredoxin Reductase Inhibitors and    Thioredoxin Inhibitors, as Measured by the Viability of Treated A431    Cells

A431 cells were treated with either one of the gold (I) thiolatesauranofin (AUR), aurothiomalate (ATM) or aurothioglucose (ATG), thealkylating agent dichloronitrobenzene (DNCB) or the thioredoxininhibitor PX12 for 24 hours with a range of concentrations of eachcompound (0.1-50 μM AUR; 0.1-500 μM ATG and ATM; 0.5-100 μM DNCB andPX12). Following treatment, cell viability was measured using aresazurin-based fluorescence assay. The mean viability of the cellsfollowing treatment was calculated as a percentage of cell viabilitycompared to the untreated controls.

The data in FIG. 25 show that both ATM and ATG were well tolerated atall concentrations tested. ATG exhibited a small (but statisticallysignificant) increase in cytotoxicity at 500 μM (95.2±4.4%, p<0.01). AURwas generally well tolerated up to a concentration of 10 μM. AURexhibited a small (but statistically significant) increase incytotoxicity at 1 (90.6±2.6%, p<0.05 c.f. untreated control) and 5 μM(87.1±2.4%, p<0.05). At 50 μM there was an almost complete loss of cellviability, with only 15.5±2.4% (p<0.001) viability compared to untreatedcontrol.

DNCB exhibited no cytotoxicity at concentrations up to 10 μM. At 50 μMand 100 μM DNCB induced significant cytotoxicity, with 11.7±0.8% (p<0.01c.f. untreated control) and 14.8±4.2% (p<0.01) viability, respectively.PX12 was also well tolerated, with no cytotoxicity observed up to aconcentration of 100 μM. At 500 μM there was a significant increase incytotoxicity, with 47.5±4.9% (p<0.01 c.f. untreated control) cellviability.

FIG. 26—A431 Cell Death Induced by Photodynamic Cell Killing FollowingTreatment with MAL in the Absence and Presence of ThioredoxinAntioxidant System Inhibitors: Thioredoxin Reductase Inhibitors andThioredoxin Inhibitors Significantly Increase MAL-Based PhotodynamicCell Killing

A431 cells were treated with MAL (1 mM) in the absence and presence ofthe thioredoxin antioxidant system inhibitors AUR (1 μM), ATM (20 μM),ATG (20 μM), DNCB (10 μM) or PX12 (10 μM) for 3 hours, after which cellswere irradiated for 5 minutes (630 nm, 25 J/cm²) and then incubated forfurther 3 hours. Cell viability was assessed by annexin V-FITC andpropidium iodide staining in conjunction with flow cytometry. Resultsare set out in FIG. 26.

The mean cell death of the untreated control group was 9.3±1.2%.Photo-irradiation of cells treated with MAL in the absence of anyinhibitor resulted in a significant increase in cell death (29.1±3.8%,p<0.001 c.f. control).

Photo-irradiation of cells co-treated of A431 cells with MAL andthioredoxin antioxidant system inhibitors lead to a significant increasein cell death compared to MAL alone. Gold (I) thiolates ATM and ATGincreased cell death to 66.7±4.9% (p<0.001 c.f. MAL alone) and 58.7±0.8%(p<0.001), respectively. AUR increased cell death to 61.3±3.3% (p<0.001)compared to MAL alone. The thioredoxin inhibitor, PX12, increased celldeath to 70.5±4.5% (p<0.001 c.f. MAL alone) and the alkylating agent,DNCB, increased cell death to 43.8±3.9% (p<0.01).

FIG. 27—Modes of A431 Cell Death Induced by Photodynamic Cell KillingFollowing Treatment with MAL in the Absence and Presence of ThioredoxinReductase Inhibitors and Thioredoxin Inhibitors: Thioredoxin ReductaseInhibitors and Thioredoxin Inhibitors Significantly Increase MAL-BasedPhotodynamic Cell Killing Through Promotion of Primarily Apoptotic CellDeath

Annexin V-FITC and propidium iodide staining was used to assess themodes of cell death following irradiation of A431 cells treated with MAL(1 mM) in the absence and presence of AUR (1 μM), ATM (20 μM), ATG (20μM), DNCB (10 μM) or PX12 (10 μM). Results are set out in FIG. 27.

Photodynamic irradiation of A431 cells pre-treated with MAL (1 mM)resulted in an increase in all types of cell death compared to theuntreated controls. Co-treatment of MAL with the thioredoxin systeminhibitors resulted in further increases in cell death, primarily lateapoptosis and necrosis, with little effect on apoptotic cell death.

FIG. 28—A431 Cell Death Induced by Photodynamic Cell Killing FollowingTreatment with MAL in the Absence and Presence of DifferentConcentrations of Auranofin (AUR): Auranofin, Another ThioredoxinReductase Inhibitor, Potentiates MAL-Induced Photodynamic Cell Killingin a Concentration-Dependent Manner

A431 cells were treated with MAL (1 mM) in the absence and presence ofdifferent concentrations of AUR (10, 100 or 1000 nM) for 3 hours, afterwhich cells were irradiated for 5 minutes (630 nm, 25 J/cm²) and thenincubated for further 3 hours. Cell viability was assessed by annexinV-FITC and propidium iodide staining in conjunction with flow cytometry.Results are set out in FIG. 28.

The mean cell death of the untreated control group was 9.3±1.2%.

The data show that treatment with MAL alone resulted in a significantincrease in cell death (29.1±3.8%, p<0.01 c.f. untreated control).Co-treatment of A431 cells with MAL and 10 nM lead to a small(non-significant) increase in cell death (35.3±4.2%, p>0.05 c.f. MALalone). Co-treatment of MAL with AUR at concentration of 100 or 1000 nMlead to statistically significant increases in cell death compared toMAL alone, with 56.7±6.3% (p<0.001) and 61.3±3.3% (p<0.001) cell deathobserved, respectively.

FIG. 29—Modes of A431 Cell Death Induced by Photodynamic Cell KillingFollowing Treatment with MAL in the Absence and Presence of DifferentConcentrations of Auranofin (AUR): Auranofin Potentiates MAL-InducedPhotodynamic Cell Killing in a Concentration-Dependent Manner ThroughPromotion of Apoptotic and Necrotic Cell Death

Annexin V-FITC and propidium iodide staining was used to assess themodes of cell death following irradiation of A431 cells treated with MAL(1 mM) in the absence and presence of AUR (10, 100 and 1000 nM). Resultsare set out in FIG. 29.

Photodynamic irradiation of A431 cells pre-treated with MAL (1 mM)resulted in an increase in all types of cell death compared to theuntreated controls. Co-treatment with AUR lead toconcentration-dependent increases in late apoptosis and necrosis, withlittle effect on early apoptosis.

FIG. 30—PpIX Accumulation in A431 Cells Following Treatment with MAL inthe Absence and Presence of Thioredoxin Antioxidant System Inhibitors:Thioredoxin Reductase Inhibitors and Thioredoxin Inhibitors Did notIncrease MAL-Induced Protoporphyrin IX Accumulation

A431 cells were treated concurrently with MAL (1 mM) in the absence andpresence of AUR (1 μM), ATM (20 μM), ATG (20 μM), DNCB (10 μM) or PX12(10 μM) for 3 hours, after which PpIX accumulation was measured byfluorescence plate reader (excitation 410 nm, excitation 630 nm). CP94(50 μM) was used as a positive control and for comparison. Results areset out in FIG. 30.

The effects of thioredoxin antioxidant system inhibitors, AUR (1 μM),ATM (20 μM), ATG (20 μM), DNCB (10 μM) and PX12 (10 μM) on PpIXaccumulation were investigated. The data presented in FIG. 30 show themeasured relative fluorescence of PpIX. MAL (1 mM) significantlyincreased PpIX accumulation compared to untreated controls (p<0.001). Insharp contrast to CP94 (an iron chelator and well-known enhancer of PpIXaccumulation used clinically), none of the thioredoxin antioxidantsystem inhibitors tested exhibited any effects on PpIX accumulation(p>0.05 c.f. 1 mM MAL) e.g. inhibition or further accumulation. CP94significantly increased PpIX accumulation compared to MAL alone(p<0.001).

FIG. 31—the Effects of Thioredoxin Antioxidant System Inhibitors onReactive Oxygen Species Generation During Photodynamic Irradiation ofA431 Cells Pre-Treated with MAL: Thioredoxin Reductase Inhibitors andThioredoxin Inhibitors Increase Mitochondrial Oxidant Generation DuringMAL-Based Photodynamic Cell Killing

A431 cells were treated concurrently with MAL (1 mM) in the absence andpresence of AUR (1 μM), ATM (20 μM), ATG (20 μM), DNCB (10 μM) or PX12(10 μM) for 3 hours and MitoSOX (2.5 μM) for 1 hour, after which theywere irradiated for 5 min (630 nm, 25 J/cm²) and MitoSOX (mito-2-OH-E⁺)fluorescence, indicative of mitochondrial oxidant production, wasimmediately analysed by flow cytometry. Results are set out in FIG. 31.

Mitochondria-targeted dihydroethidium was used to assess the productionof reactive oxygen species (ROS) by PpIX photochemical reactions and todetermine the effects of thioredoxin reductase inhibitors andthioredoxin inhibitors on this process. All of the results arerepresented as a percentage of untreated cells (100.0±4.3%).

The fluorescence of the photoirradiated cells, which had beenpre-treated with MAL alone, was 143.3±4.8% (p<0.001 c.f. untreatedcontrols). Co-treatment of MAL with AUR (1 μM) resulted in a furtherincrease in MitoSOX fluorescence to 182.6±8.3% (p<0.001, c.f. MALalone). ATM and ATG also significantly increased MitoSOX fluorescence to192.4±5.6% (p<0.001) and 184.9±5.9% (p<0.001), respectively.

Co-treatment of MAL with DNCB also increased MitoSOX fluorescence to209.5±3.2% (p<0.001, c.f. MAL alone) and PX12 increased fluorescence to211.4±17.6% (p<0.001).

EXAMPLE 3 Effects of Nitroxides on Photodynamic Cell Killing

Compounds Tested

The effects of nitroxides on photodynamic cell killing were investigatedby using the nitroxides TEMPO and TEMPOL, which have the followingstructures:

TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) is commercially availablefrom Sigma Aldrich and Enzo Life Sciences.

TEMPOL (4-hydroxy-2,2,6,6-tetra methylpiperidine 1-oxyl) is commerciallyavailable from Sigma Aldrich and Enzo Life Sciences.

Toxicity of Nitroxides TEMPO and TEMPOL

A431 cells were treated with 1 mM MAL±TEMPOL or TEMPO (0-10 mM) for 3hours, after which cell viability was measured using a resazurin-basedfluorescence assay (FIG. 11). The mean viability of cells followingtreatment was calculated as a percentage of viability compared to theuntreated control cells (TEMPOL—100.0±5.5% and TEMPO—100.0±3.1%).

Treatment with TEMPOL at concentrations of 0.25, 0.5, 1 or 2 mM did notsignificantly affect cell viability, with 99.3±4.4%, 102.0±3.6%,99.8±2.9% and 104.2±3.3%, respectively. Similarly, viability of cellstreated with TEMPO over the same range was 97.8±3.4%, 97.5±1.7%,98.6±6.8% and 97.4±6.2%, respectively.

Treatment with 5 mM TEMPOL resulted in a statistically significantincrease in viability, to 116.2±2.7% (p<0.01), whilst 10 mM resulted insignificant decrease in viability, to 77.8±4.7% (p<0.001). Treatmentwith 5 and 10 mM TEMPO led to significant decreases in viability, with92.9±6.6% (p<0.05) and 79.9±8.5% (p<0.01), respectively.

The Effects of Co-Treatment with Nitroxides TEMPO or TEMPOL on MAL-BasedPhotodynamic Cell Killing

A431 cells were treated with 1 mM MAL in absence and presence of TEMPO(1 mM) or TEMPOL (1 mM) for 3 h, after which cells were irradiated for 5min (630 nm, 25 J/cm²) and then incubated for further 3 h, so A431 cellviability was assessed 3 hours post-irradiation. Viability was assessedby annexin V-FITC and propidium iodide staining in conjunction with flowcytometry.

Measurements of total cell death following treatment of the cells withMAL in the absence and presence of TEMPO or TEMPOL have been plotted inFIG. 12. The mean cell death of the untreated control group was10.7±2.3%.

Treatment with 1 mM MAL and irradiation alone (in the absence ofco-treatment) resulted in 34.4±3.2% cell death; a statisticallysignificant increase compared to untreated control (p<0.001).

Co-treatment with 1 mM MAL, 1 mM TEMPO or 1 mM TEMPOL and irradiationresulted in 52.0±8.3% and 50.8±6.8% cell death, respectively. Theseincreases in cell death were statistically significant compared tountreated control (p<0.001) and 1 mM MAL and irradiation (p<0.001).

A breakdown of A431 cell death for each treatment has been plotted inFIG. 13. Annexin V-FITC and propidium iodide staining was used to assessthe modes of cell death.

Treatment with MAL and irradiation resulted in an increase in all typesof cell death compared to untreated controls.

Co-treatment with TEMPO resulted in an increase in apoptotic and lateapoptotic cell death, whilst co-treatment with TEMPOL resulted in anincrease in late apoptotic and necrotic cell death.

Further experiments were carried out to investigate the effects ofTEMPOL and TEMPO on MAL-induced PpIX accumulation. A431 cells weretreated with 1 mM MAL in absence and presence of TEMPOL or TEMPO (0.01-1mM) for 3 h, after which PpIX accumulation was measured by fluorescenceplate reader (excitation 410 nm, excitation 630 nm). FIG. 3.17 shows themeasured relative fluorescence of PpIX. As the concentration of TEMPOLor TEMPO was increased, a corresponding increase in PpIX fluorescencewas detected. TEMPOL and TEMPO at 0.01 mM increased PpIX fluorescenceslightly, but this was not statistically significant. At 0.1 mM and 1mM, TEMPOL significantly increased PpIX accumulation further (p<0.05 andp<0.001, respectively). Similar results were observed with TEMPOL at 0.1mM and 1 mM (p<0.05 and p<0.01, respectively). With both antioxidants,addition of 1 mM resulted in a ˜2-fold increase in PpIX fluorescence.

EXAMPLE 3A Nitroxides Potentiate Methyl-Aminolaevulinic Acid-BasedPhotodynamic Cell Killing

The data covered in this Example builds on the previously identifiedabilities of a class of stable radicals, known as nitroxides, topotentiate methyl-aminolaevulinic acid (MAL; Metvix®) photodynamic cellkilling.

In short, these data show that several nitroxides are well toleratedacross a large range of concentrations and only begin to exhibitcytotoxicity outside of the “therapeutic” concentration. This toleranceis exhibited over periods relevant to the photodynamic experimentscarried out (24 hours), suggesting the compounds are safe at theconcentrations carried forward into further investigation.

TEMPONE exhibits a mild potentiating effect, whilst TEMPO and TEMPOLexhibit a more robust potentiating effect. The observed increases intotal cell death were driven by increases in late apoptosis andnecrosis, with little-to-no effect on early apoptosis. Themitochondria-targeted nitroxide, MitoTEMPO also exhibited a potentiatingeffect, equivalent to the non-targeted TEMPO at a concentration 20-foldlower. Further investigation with TEMPOL revealed that thesepotentiating effects are concentration-dependent.

By measuring the effects of these compounds on MAL-inducedprotoporphyrin IX (PpIX) accumulation, it has been established that alltested nitroxides significantly increased PpIX accumulation in aconcentration-dependent manner. Previous work has highlighted that thiseffect is not elicited through iron chelation (which is how CP94 exertsits effects), but rather through iron oxidation. MitoTEMPO was alsofound to be 20-fold more potent than TEMPO at increasing PpIXaccumulation (to equivalent levels), supporting observations made whenmeasuring effects on photodynamic cell killing.

Significant increases in mitochondrial oxidant generation were detectedfollowing photodynamic irradiation of cells co-treated with MAL and eachof the nitroxides.

Further investigation with TEMPOL revealed that this increase in oxidantgeneration appears to occur in a concentration-dependent manner.

Whilst the potentiating effect of these nitroxides is observed atconcentrations greater than CP94 (50 μM) which exerts a similar degreeof potentiation, this work has established a novel mechanism by whichthe efficacy of PpIX-based photodynamic cell killing could be increased.Furthermore, we have established that targeting a nitroxide to themitochondria is a valid method by which potency can be significantlyincreased.

FIG. 32—Dark Toxicity of Nitroxides, as Measured by the Viability ofTreated A431 Cells

A431 cells were treated with the nitroxides TEMPOL, TEMPONE, TEMPO andits mitochondria-targeted derivative, MitoTEMPO for 24 hours with arange of concentrations of each compound (0.1-50 mM TEMPOL, TEMPONE,TEMPONE; 0.5-500 μM MitoTEMPO), after which cell viability was measuredusing a resazurin-based fluorescence assay. The mean viability of thecells following treatment was calculated as a percentage of cellviability compared to the untreated controls.

The data in FIG. 32 show that treatment with MitoTEMPO was welltolerated up to a concentration of 100 μM. At 500 μM, a small butstatistically significant decrease in cell viability was observed(86.5±1.6%, p<0.01 c.f. control). TEMPONE and TEMPO did not exhibit anycytotoxicity up to a concentration of 5 mM. At higher concentrations,TEMPONE and TEMPO showed significant cytotoxicity, with 80.3±13.3%(p=0.05 c.f. control) and 64.9±0.1% (p<0.01) of untreated cells at 10 mMand 53.7±13.3% (p=0.01) and 8.5±0.2% (p<0.001) of untreated cells at 50mM, respectively.

FIG. 33—A431 Cell Death Induced by Photodynamic Cell Killing FollowingTreatment with MAL in the Absence and Presence of Nitroxides: NitroxidesSignificantly Increase MAL-Based Photodynamic Cell Killing

A431 cells were treated with 1 mM MAL in the absence and presence ofTEMPOL (1 mM) TEMPONE (1 mM), TEMPO (1 mM) or MitoTEMPO (50 μM) for 3hours, after which cells were irradiated for 5 minutes (630 nm, 25J/cm²) and then incubated for further 3 hours. Cell viability wasassessed by annexin V-FITC and propidium iodide staining in conjunctionwith flow cytometry. Results are set out in FIG. 33.

The mean cell death of the untreated control group was 11.7±3.4%.Treatment with 1 mM MAL in the absence of any nitroxide resulted in asignificant increase in cell death (26.8±1.9%, p<0.001 c.f. control).Co-treatment of A431 cells with MAL and TEMPOL, TEMPONE or TEMPO lead tostatistically significant increases in cell death compared to MAL alone,with 69.3±3.1% (p<0.001), 37.9±2.3% (p<0.001) and 46.3±3.6% (p<0.001)cell death observed, respectively. Co-treatment with MitoTEMPO also leadto a statistically significant increase in cell death (43.3±7.7%,p<0.01) compared to MAL alone.

FIG. 34—Modes of A431 Cell Death Induced by Photodynamic Cell KillingFollowing Treatment with MAL in the Absence and Presence of Nitroxides:Nitroxides Significantly Increase MAL-Based Photodynamic Cell KillingThrough Promotion of Apoptotic and Necrotic Cell Death

Annexin V-FITC and propidium iodide staining was used to assess themodes of cell death following irradiation of A431 cells treated with MAL(1 mM) in absence and presence of the nitroxides TEMPOL (1 mM), TEMPONE(1 mM), TEMPO (1 mM) and MitoTEMPO (50 μM). Results are set out in FIG.34.

Photodynamic irradiation of A431 cells pre-treated with 1 mM MALresulted in an increase in all types of cell death compared to theuntreated controls. Co-treatment with TEMPOL, TEMPONE, TEMPO orMitoTEMPO resulted in a further increase in late apoptosis and necrosiswith little effect on apoptotic cell death.

FIG. 35—Concentration-Response Plot Showing the Effects of TEMPO andMitoTEMPO on MAL-Induced PpIX Accumulation in A431 Cells: TheMitochondria-Targeted MitoTEMPO is Significantly More Potent atIncreasing MAL-Induced PpIX than its Non-Targeted Parent Compound, TEMPO

A431 cells were treated concurrently with MAL (1 mM) and TEMPO (50-5000μM), MitoTEMPO (0.1-100 μM) or CP94 (0.1-100 μM) for 3 hours. Followingtreatment, PpIX accumulation was measured by fluorescence plate reader(excitation 410 nm, excitation 630 nm).

The effects of TEMPO and MitoTEMPO on MAL-induced PpIX accumulation wereinvestigated. CP94, a well-known enhancer of PpIX accumulation, was usedas a positive control and for comparison. The data presented in FIG. 35show the measured increase in relative fluorescence of PpIX, compared totreatment with MAL (1 mM) alone.

The data show that each compound increased PpIX accumulation in A431cells in a typical concentration-response manner. MitoTEMPO issignificantly more potent than TEMPO at increasing MAL-induced PpIXaccumulation, but less so than CP94. For example, at 50 μM TEMPO did nothave any statistically significant effect (0.0±4.9%), whilst MitoTEMPOinduced a 109.1±9.6% increase in PpIX accumulation (p<0.001 c.f. 50 μMTEMPO) and CP94 increased PpIX by 223.2±26.1% (p=0.001 c.f. 50 μMMitoTEMPO).

The peak response for TEMPO was obtained at 5 mM (138.1±10.5%) and apeak response for MitoTEMPO was obtained at 100 μM (126.0±11.8%), a50-fold lower concentration. The peak response for CP94 was alsoobtained at 100 μM, but this was significantly higher than the responsefrom MitoTEMPO (228.1±42.3% p<0.001).

FIG. 36—A431 Cell Death Induced by Photodynamic Cell Killing FollowingTreatment with MAL in the Absence and Presence of DifferentConcentrations of TEMPOL: TEMPOL Potentiates MAL-Induced PhotodynamicCell Killing in a Concentration-Dependent Manner

A431 cells were treated with MAL (1 mM) in the absence and presence ofTEMPOL (10, 100 and 1000 μM) for 3 hours, after which cells wereirradiated for 5 minutes (630 nm, 25 J/cm²) and then incubated forfurther 3 hours. Cell viability was assessed by annexin V-FITC andpropidium iodide staining in conjunction with flow cytometry.

The mean cell death of the untreated control group was 13.4±1.5%.

The data in FIG. 36 show that treatment with MAL alone resulted in asignificant increase in cell death (24.6±4.2%, p<0.01 c.f. control).Co-treatment of A431 cells with MAL and 10, 100 or 1000 μM TEMPOL leadto statistically significant increases in cell death (in aconcentration-dependent manner) compared to MAL alone, with 40.5±4.1%(p<0.001), 48.5±7.4% (p<0.001) and 69.3±3.1% (p<0.001) cell deathobserved, respectively.

FIG. 37—Modes of A431 Cell Death Induced by Photodynamic Cell KillingFollowing Treatment with MAL in the Absence and Presence of Nitroxides:TEMPOL Potentiates MAL-Induced Photodynamic Cell Killing in aConcentration-Dependent Manner Through Promotion of Apoptotic andNecrotic Cell Death

Annexin V-FITC and propidium iodide staining was used to assess themodes of cell death following irradiation of A431 cells treated with MAL(1 mM) in absence and presence of the nitroxides TEMPOL (1 mM), TEMPONE(1 mM), TEMPO (1 mM) and MitoTEMPO (50 μM). Results are set out in FIG.37.

Photodynamic irradiation of A431 cells pre-treated with MAL (1 mM)resulted in an increase in all types of cell death compared to theuntreated controls. Co-treatment with TEMPOL lead to an increase inapoptosis and late apoptosis, with smaller increases in necrosis alsoobserved. These increases were dependent on the concentration of TEMPOL.

FIG. 38—Concentration-Response Plot Showing the Effects of TEMPOL,TEMPONE and TEMPO on MAL-Induced PpIX Accumulation in A431 Cells:TEMPOL, TEMPONE and TEMPO Increase MAL-Induced PpIX Accumulation in aConcentration-Dependent Manner

A431 cells were treated concurrently with MAL (1 mM) and either TEMPOL,TEMPONE or TEMPO (10, 100 or 1000 μM) for 3 hours. Following treatment,PpIX accumulation was measured by fluorescence plate reader (excitation410 nm, excitation 630 nm).

The effects of TEMPOL, TEMPONE and TEMPO on MAL-induced PpIXaccumulation were investigated. The data in FIG. 38 are presented as apercentage of the MAL alone treatment (100±2.8).

PpIX accumulation was significantly increased by TEMPOL at 10, 100 and1000 μM, with 109.0±2.7% (p<0.01 c.f. MAL alone), 141.2±4.2% (p<0.001)and 209.2±5.1% (p<0.001) PpIX accumulation, respectively.

At 10 uM, neither TEMPO (100.3±3.8%) nor TEMPONE (100.3±4.0%) had anyeffect on PpIX accumulation (p>0.05). Statistically significantincreases were induced by TEMPO at 100 and 1000 μM, with 127.0±3.3%(p<0.001 c.f. MAL alone) and 159.2±4.1% (p<0.001 c.f. MAL alone) PpIXobserved, respectively. TEMPONE had similar effects, with 123.8±2.5% and160.2±3.2% PpIX at 100 and 1000 μM, respectively.

FIG. 39—the Effects of Nitroxides on Reactive Oxygen Species GenerationDuring Photodynamic Irradiation of A431 Cells Pre-Treated with MAL:Nitroxides Increase Mitochondrial Oxidant Generation During MAL-BasedPhotodynamic Cell Killing

A431 cells were treated concurrently with TEMPOL, TEMPONE, TEMPO (1 mM),MitoTEMPO or CP94 (50 μM) and MAL (1 mM) for 3 hours and MitoSOX (2.5μM) for 1 hour, after which they were irradiated for 5 min (630 nm, 25J/cm²) and MitoSOX (mito-2-OH-E⁺) fluorescence, indicative ofmitochondrial oxidant production, was immediately analysed by flowcytometry. Results are set out in FIG. 39.

Mitochondria-targeted dihydroethidium was used to assess the productionof reactive oxygen species (ROS) by PpIX photochemical reactions and todetermine the effects of nitroxides on this process. All of the resultsare represented as a percentage of untreated cells (100.0±4.3%).

The fluorescence of the photoirradiated cells, which had beenpre-treated with MAL alone, was 143.3±4.8% (p<0.001 c.f. untreatedcontrols). Co-treatment of MAL with CP94 or MitoTEMPO (50 μM) resultedin a further increase in MitoSOX fluorescence to 198.4±7.7% (p<0.001,c.f. MAL alone) and 220.3±9.0% (p<0.001), respectively. Co-treatment ofMAL with TEMPOL, TEMPONE and TEMPO (1 mM) also increased MitoSOXfluorescence to 253.3±6.3% (p<0.001, c.f. MAL alone), 226.4±6.8%(p<0.001) and 418.8±6.3% (p<0.001), respectively.

FIG. 40—the Effects of Different Concentrations of TEMPOL on ReactiveOxygen Species Generation During Photodynamic Irradiation of A431 CellsPre-Treated with MAL TEMPOL Increases Mitochondrial Oxidant GenerationDuring MAL-Based Photodynamic Cell Killing in a Concentration-DependentManner

A431 cells were treated concurrently with TEMPOL (10, 100 or 1000 μM)and MAL (1 mM) for 3 hours and MitoSOX (2.5 μM) for 1 hour, after whichthey were irradiated for 5 min (630 nm, 25 J/cm²) and MitoSOX(mito-2-OH-E⁺) fluorescence, indicative of mitochondrial oxidantproduction, was immediately analysed by flow cytometry. Results are setout in FIG. 40.

Mitochondria-targeted dihydroethidium was used to assess the productionof reactive oxygen species (ROS) by PpIX photochemical reactions and todetermine the effects of nitroxides on this process. All of the resultsare represented as a percentage of untreated cells (100.0±5.8%).

The fluorescence of the photoirradiated cells, which had beenpre-treated with MAL alone, was 207.4±16.7% (p<0.001 c.f. untreatedcontrols). Co-treatment of MAL with 10 μM TEMPOL had no statisticallysignificant effect on MitoSOX fluorescence (99.4±5.44% p>0.05 c.f. MALalone). Co-treatment of MAL with 100 and 1000 μM TEMPOL significantlyincreased MitoSOX fluorescence, compared to MAL alone, with 226.5±19.4%(p<0.05) and 316.8±1.6% (p<0.001) fluorescence, respectively.

The invention claimed is:
 1. A combination comprising: (i) an inhibitorof the thioredoxin antioxidant system selected from the group consistingof auranofin, aurothiomalate, aurothiosulfate, aurothioglucose, DNCB andPX12; and (ii) a photosensitizer or photosensitizer precursor selectedfrom the group consisting aminolaevulinic acid (ALA), methylaminolaevulinate (MAL), hexyl aminolaevulinate (HAL), and a combinationthereof; for use in photodynamic therapy.
 2. The combination accordingto claim 1 wherein the inhibitor is auranofin.
 3. The combinationaccording to claim 1, wherein the inhibitor is DNCB.
 4. The combinationfor use according to claim 1, wherein the inhibitor is PX12.
 5. Thecombination for use according to claim 1, wherein the photosensitizerprecursor is methyl aminolaevulinate (MAL).
 6. A method of treatingcancer, comprising administering to a patient in need thereof: aphotodynamic therapy with a combination set forth in claim
 1. 7. Amethod of treating a condition, comprising administering to a patient inneed thereof: a photodynamic therapy with a combination set forth inclaim 1, wherein said condition is selected from the group consisting ofscleroderma, lichen sclerosus, psoriasis, warts, chronic wounds, acne, amicrobial infection, a parasitic infestation, rheumatoid arthritis orleukaemia.
 8. A method of purging bone marrow, comprising administeringto a patient in need thereof: a photodynamic therapy with a combinationset forth in claim 1.